Fatty acid composition and change in Collembola fed differing diets:
identification of trophic biomarkers
P.M. Chamberlaina,b, I.D. Bulla, H.I.J. Blackb, P. Inesonc, R.P. Eversheda,*
aOrganic Geochemistry Unit, Bristol Biogeochemistry Research Centre, School of Chemistry, University of Bristol, Cantock’s Close, Bristol BS8 1TS, UKbCentre for Ecology and Hydrology, Lancaster Environment Centre, Library Avenue, Bailrigg, Lancaster LA1 4AP, UK
cDepartment of Biology, University of York, PO Box 373, York YO10 5WY, UK
Received 5 December 2003; received in revised form 24 November 2004; accepted 27 January 2005
Abstract
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.
q 2005 Elsevier Ltd. All rights reserved.
Keywords: Diet; Biomarker; Fatty acids; Polyunsaturated; Collembola
1. Introduction
The use of fatty acids (FAs) as trophic biomarkers is
common in aquatic ecosystems (e.g. Nelson et al., 2001;
Auel et al., 2002), but their potential use in soils, for
example in soil food web studies, has not been well
explored. Amongst soil organisms, the FA compositions of
bacteria and fungi have been extensively investigated (e.g.
Zelles, 1999; Madan et al., 2002; Baath, 2003), as have
the FA compositions of nematodes (e.g. Chitwood and
Krusberg, 1981; Tanaka et al., 1996), and there has been
limited, fundamental, research into the FAs of enchytraeids
(Jacob et al., 1991), slugs and snails (Weinert et al., 1993;
Zhu et al., 1994) and earthworms (e.g. Albro et al., 1992;
0038-0717/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.soilbio.2005.01.022
* Corresponding author. Tel.: C44 117 9287674; fax: C44 117 9251295.
E-mail address: [email protected] (R.P. Evershed).
Petersen and Holmstrup, 2000). However, there has been
very little study of the FAs of microarthropods such as
Collembola (Stransky et al., 1986; Holmstrup et al., 2002;
Haubert et al., 2004; Ruess et al., 2004) and mites, which are
significant and common components of the soil biological
community (Blakely et al., 2002). Fewer investigators still
have examined the changes in the FA compositions of soil
organisms over time (rare exceptions being Chen et al.
(2001) and Haubert et al. (2004)), or the rates of turnover
of FAs in such organisms. The analytical challenge of
determining the composition of the small quantities of FAs
present in soil organisms, such as the microarthropods, has
also been a hurdle resulting in the lack of previous work.
Fatty acids occur in many forms in organisms. Phospho-
lipid fatty acids (PLFAs) are structural components of cell
membranes, while neutral lipid fatty acids (NLFAs) in
triacylglycerols act as a store of energy and are often the
dominant form in which FAs are found in insects
(Stanley-Samuelson et al., 1988). Fatty acids may also
occur as free components or bound in glycolipids and steryl
Soil Biology & Biochemistry 37 (2005) 1608–1624
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P.M. Chamberlain et al. / Soil Biology & Biochemistry 37 (2005) 1608–1624 1609
fatty acyl esters. Among invertebrates, the focus has been
largely on commercially-important, above-ground
insects and a great deal is known about the composition of
insect FAs which have been shown to vary between species
(Sushchik et al., 2003), life-stage (Ogg and Stanley-Sa-
muelson, 1992), environment (e.g. Joanisse and Storey,
1996) and diet (Geer and Perille, 1977; Hanson et al., 1985).
PLFA compositions change in response to temperature and
moisture conditions (Hazel and Williams, 1990) to ensure
the required fluidity of cell membranes while the relative
abundance of NLFAs are inevitably reduced when an
invertebrate is starved (Renault et al., 2002; Haubert et al.,
2004).
Amongst soil organisms, nematode FAs are known to
change in response to temperature and other conditions
(Tanaka et al., 1996; Abu Hatab and Gaugler, 2001) and the
FA compositions of nematodes are responsive to the FA
compositions of their diet (Abu Hatab et al., 1998; Ruess
et al., 2002). However, there have been few examinations of
the effects of diet on Collembolan FAs (Haubert et al., 2004;
Ruess et al., 2004) despite the wide range of diets (and
therefore FAs) Collembola are known to consume includ-
ing: plant detritus (Takeda and Ichimura, 1983; Ponge,
2000), algae (Vegter, 1983), fungi (e.g. Sadaka-Laulan
et al., 1998), nematodes (Lee and Widden, 1996) and pollen
(Kato, 1995).
In an experiment designed to examine the effects of
different diets on Collembolan total FA compositions with
time, we fed two species of Collembola, Folsomia candida
and Proisotoma minuta, four diets representative of various
components of the soil ecosystem which Collembola
may regularly ingest. The FA compositions of the
Collembola were then examined over the ensuing 39 days.
We hypothesised that: (i) Collembolan FA profiles would
change in response to diet, (ii) that certain dietary FAs
would be directly incorporated into Collembolan FAs, and
(iii) that some of these would therefore have potential value
as trophic biomarkers.
2. Materials and methods
2.1. Experimental design
Diet-switches were carried out in a replicated experiment
with microcosms containing either F. candida (Willem) or
P. minuta (Tullberg) and one of four different foodstuffs:
Cladosporium cladosporioides (Frensen.), Panagrellus
redivivus (Linne, 1767), Zea mays L. (maize) and Alnus
glutinosa L. (alder) leaves.
C. cladosporioides was produced using a modified
Czapek Dox culturing technique with sucrose as the sole
C source. Sucrose (30 g), NaNO3 (3 g), KH2PO4 (1 g),
MgSO4 (0.5 g), KCl (0.5 g) and Fe2SO4 (0.01 g) were
dissolved in 1 l distilled water, autoclaved (15 min at
121 8C) and allowed to cool before inoculation with
C. cladosporioides obtained from the fungal collection
held at Merlewood Research Station, Cumbria, UK. The
inoculated media was shaken for 14 days after which the
fungus was filtered onto glass fibre filter paper (Whatman)
and stored in a Petri dish at 4 8C.
P. redivivus was grown on a substrate of maize meal
(Dunns River Fine Cornmeal, Enco Products Ltd, Hatfield,
Herts, UK). The meal was mixed with double distilled water
(100 ml H2O 100 gK1 maize meal) before being placed in
500 ml glass jars and autoclaved (15 min). After the
substrate had cooled, it was inoculated with P. redivivus
using aseptic techniques. The jars were kept in the dark at
20 8C until required. Nematodes were extracted from the
maize matrix by filtering a solution of distilled water,
nematodes and maize meal through a nylon mesh (90 mm).
The resulting suspension of nematodes was collected from
the bottom of the filter funnel. The nematodes were further
concentrated by centrifugation and excess water removed;
the resultant nematode slurry was added to the experimental
microcosms by pipette.
Maize litter was obtained from Sizergh Farm, near
Kendal, Cumbria, UK, where it was harvested as a fodder
crop. The maize was collected as chopped material prepared
for silage and then air-dried. Alder leaves were collected
from the Gisburn Forest Experiment, Yorkshire, UK and air-
dried for storage. Before use, both plant litters were soaked
overnight in distilled water and then thoroughly rinsed.
F. candida and P. minuta were taken from stock colonies
reared on bakers yeast (Saccharomyces cerevisiae) and
placed in groups of 20 into individual microcosms
comprising small plastic pots (38!65 mm) with a plaster
of Paris and charcoal base (Snider, 1972). The plaster of
Paris bases were soaked in distilled water for 24 h prior to
introduction of the Collembola in order to maintain a high
relative humidity in the microcosms throughout the
experiment. In all cases the Collembola were observed to
be feeding directly on the foodstuffs offered to them.
Microcosms were destructively sampled on days 2, 4, 8, 16
and 39. Collembola from the stock colonies were taken as
day 0 samples. Three replicates of each diet were taken at
each sampling point. Collembola were removed from the
food sources for 2 h prior to freezing to allow complete
passage of ingested foods from the gut.
2.2. Lipid characterisation
Lipids were extracted and analysed as described in
Chamberlain et al. (2004). Briefly, freeze-dried Collembola
and diets were lipid extracted with 2:1 DCM/methanol
(3!2 ml). Lipids were saponified with 0.5 M NaOH in
methanol (70 8C for 90 min), the solution acidified with
0.5 M HCl and the lipids extracted into DCM. Fatty acids
were separated from other lipids on an aminopropyl solid
phase extraction cartridge (Bond Elut NH2, Varian) using
DCM/2-propanol (2:1 v/v) to elute the neutral fraction,
and diethyl ether containing 2% acetic acid to elute the
P.M. Chamberlain et al. / Soil Biology & Biochemistry 37 (2005) 1608–16241610
FAs. Fatty acids were derivatised using boron trifluoride-
methanol (14% w/v, 30 ml, 70 8C, 10 min) to produce fatty
acid methyl esters (FAMEs). The FAME compositions
were determined by gas chromatography (GC) and
compounds identified using gas chromatography/mass
spectrometry (GC/MS). Analytical conditions are described
in detail in Chamberlain et al. (2004).
2.3. Statistical analyses
The absolute concentration of FAs in diets and
Collembola were not determined in this study; FA
compositions are reported as a percentage of total FAs,
commonly referred to as relative abundance. Prior to
statistical analysis FA compositions were arcsine trans-
formed to normalise the distributions. All statistical
analyses were carried out using Statistica (v6.1; StatSoft,
Inc.). Repeated measures analysis of variance (ANOVA)
was used on a single dataset consisting of all Collembolan
relative abundance data to test for significant changes in FA
compositions over time; Tukeys honest significant differ-
ence test was used to determine the significance of changes
in individual components. Changes in the ratios of odd-to-
even chain length FAs and saturated-to-unsaturated FAs
were also analysed using repeated measures ANOVA, with
a post-hoc Tukey test to examine significant changes in
individual Collembola species and diets. Statistica was also
Fig. 1. Partial gas chromatograms of the fatty acid compositions of the four diets; (a
acids are: A 12:0, B 13:0, C 14:0, D 14:1, E i15:0, F a15:0, G 15:0, H i16:0, I 16:0
R 18:1(n-9), S 18:1(n-7), T 18:1(n-5), U 18:2(n-6), V 18:3, W 18:30, X 20:0, Y 20
q 23:0, k 24:0.
used for principle components analysis of the FA
compositions.
3. Results
3.1. Fatty acid compositions of offered diets
The FA compositions of the offered diets are summarised
in Fig. 1 and Tables 1–4. C. cladosporioides (Fig. 1a;
Table 1) exhibited the narrowest range of FAs, containing 7
detectable components, with a composition dominated by
16:0, 18:1(n-9) and 18:2(n-6). P. redivivus (Fig. 1b; Table 2)
contained a particularly wide range of FAs, 28 in total,
including six C20 polyunsaturated fatty acids (PUFAs)
which accounted for 24% of the total FA composition.
Maize (Fig. 1c; Table 3) contained 12 detectable FAs and
like C. cladosporioides, was dominated by 16:0, 18:1 (n-9)
and 18:2(n-6). The latter component alone accounted for
43% of the total FA composition of maize. Alder (Fig. 1d;
Table 4) contained 18 FAs, the most abundant of
which were 16:0 and 22:0. There were significant
differences in the ratios of saturated-to-unsaturated FAs of
the diets (P!0.005). C. cladosporioides, P. redivivus and
maize all exhibited ratios of saturated-to-unsaturated FAs in
the range 0.32–0.41; while the ratio of saturated-to-
unsaturated FAs in alder (2.32) was significantly higher
) C. cladosporioides, (b) P. redivivus, (c) Z. mays and (d) A. glutinosa. Fatty
, J 16:1(n-7), K 16:1(n-5), L 16:2, M i17:0, N 17:0, O 17:1, P i18:0, Q 18:0,
:1, Z 20:2(n-6), a 20:3(n-6), b 20:4(n-6), g 20:3, d 20:4, 3 20:5(n-3), z 22:0,
Table 1
Fatty acid compositions of F. candida and P. minuta fed C. cladosporioides for 39 days from day 0 of the diet-switching experiment
Fatty acid Time (days) Diet
F. candida P. minuta
0 2 4 8 16 39 0 2 4 8 16 39
16:0 12.5 (0.5) 15.1 (0.1) 16.0 (2.6) 17.7 (1.2) 14.4 (3.2) 12.0 (0.5) 15.6 (0.6) 19.2 (1.4) 16.2 (2.9) 20.9 (1.6) 18.7 (1.0) 18.4 (0.5) 18.6 (1.1)
16:1(n-7) 15.6 (1.7) 16.2 (0.7) 11.0 (0.9) 9.2 (0.8) 5.7 (0.5) 2.5 (0.1) 11.6 (2.0) 8.7 (1.3) 6.5 (0.9) 5.8 (0.5) 4.1 (0.5) 3.6 (0.6) 1.1 (0.1)
18:0 13.2 (1.0) 12.5 (0.6) 12.1 (1.6) 14.7 (0.7) 12.7 (3.3) 10.5 (1.3) 18.8 (1.9) 18.8 (0.3) 14.9 (3.2) 18.1 (0.8) 21.2 (0.4) 21.4 (0.7) 5.8 (0.4)
18:1(n-9) 35.9 (0.4) 42.2 (0.3) 43.5 (2.9) 46.4 (0.6) 41.6 (5.4) 36.7 (1.1) 38.0 (1.7) 35.8 (6.1) 37.6 (5.5) 44.8 (1.9) 47.4 (0.9) 44.6 (1.4) 36.8 (0.6)
18:1(n-7) 5.7 (0.2) 5.3 (0.1) 3.8 (0.2) 3.5 (0.2) 2.2 (0.3) 0.9 (0.6) 1.0 (0.2) 1.8 (0.2) 1.8 (0.5) 1.6 (0.1) 1.5 (0.1) 1.5 (0.2) 0.6 (0.1)
18:2(n-6) 1.7 (0.2) 2.5 (0.1) 4.5 (3.1) 1.5 (1.1) 9.7 (6.0) 14.7 (2.0) 4.5 (0.1) 5.7 (3.5) 10.3 (5.5) 3.0 (0.4) 3.1 (0.3) 3.5 (0.9) 31.9 (0.3)
18:3 0 0 0 0 1.1 (1.0) 2.5 (0.4) 0 0 0 0 0 0 4.2 (0.1)
20:4(n-6) 6.0 (1.9) 0 1.8 (3.0) 0 3.7 (3.2) 7.9 (0.2) 8.4 (0.7) 2.4 (4.1) 5.7 (5.0) 0 0 0 0
20:5(n-3) 4.2 (0.3) 0 1.0 (1.8) 0 2.1 (1.8) 4.7 (0.1) 2.1 (0.4) 0.5 (0.8) (0.8) 0 0 0 0
Total C20
PUFAs
10.2 (1.7) 0.4 (0.4) 3.0 (5.2) 0 6.4 (5.5) 14.1 (0.3) 10.1 (1.0) 2.8 (4.9) 6.6 (5.8) 0 0 0 0
Total no. of
fatty acids
19 20 23 21 25 23 15 17 19 15 13 15 7
Sat/unsat
ratioa
0.42 (0.02) 0.46 (0.01) 0.47 (0.10) 0.58 (0.04) 0.47 (0.13) 0.35 (0.01) 0.53 (0.03) 0.73 (0.09) 0.56 (0.14) 0.78 (0.05) 0.76 (0.03) 0.83 (0.04) 0.33 (0.01)
Odd/even
ratiob
0.025
(0.002)
0.027
(0.003)
0.027
(0.005)
0.034
(0.005)
0.029
(0.008)
0.027
(0.003)
0.015
(0.002)
0.026
(0.002)
0.029
(0.004)
0.039
(0.009)
0.031
(0.006)
0.048
(0.004)
0.005
(0.001)
Fatty acid compositions are mean percentages of total FAs (SD) and nZ3. All components present at O2% abundance during the experiment are shown.a Ratio of saturated-to-unsaturated fatty acids.b Ratio of odd-to-even carbon number fatty acids.
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Table 2
Fatty acid compositions of F. candida and P. minuta fed P. redivivus for 39 days from day 0 of the diet-switching experiment
Fatty acid Time (days) Diet
F. candida P. minuta
0 2 4 8 16 39 0 2 4 8 16 39
i15:0 0.5 (0.1) 2.8 (0.4) 3.4 (0.1) 5.9 (1.3) 5.2 (0.1) 6.0 (0.2) 0 1.5 (0.8) 1.9 (0.6) 3.3 (0.4) 4.4 (0.6) 5.5 (0.2) 8.2 (0.3)
16:0 12.5 (0.5) 10.2 (1.0) 8.6 (0.3) 8.7 (2.0) 6.7 (0.4) 4.6 (0.4) 14.6 (0.6) 9.6 (0.7) 8.4 (0.5) 7.9 (0.4) 8.2 (0.2) 5.4 (0.2) 2.7 (0.1)
16:1(n-7) 15.6 (1.7) 8.3 (2.0) 6.1 (0.2) 5.1 (1.0) 3.4 (0.2) 2.6 (0.2) 10.8 (2.0) 7.1 (1.8) 4.9 (0.5) 3.4 (0.1) 2.6 (0.2) 1.9 (0.1) 1.6 (0.1)
i17:0 0.7 (0.1) 1.3 (0.2) 1.8 (0.1) 2.9 (0.8) 2.3 (0.1) 2.8 (0.1) 0 1.4 (0.1) 2.0 (0.1) 2.9 (0.2) 3.8 (0.2) 4.8 (0.1) 5.2 (0.2)
18:0 13.2 (1.0) 9.9 (0.5) 10.3 (0.9) 13.5 (3.5) 11.1 (0.5) 10.0 (0.5) 17.6 (1.8) 13.1 (2.2) 14.0 (0.3) 14.0 (0.8) 14.9 (0.2) 11.3 (0.1) 6.4 (0.1)
18:1(n-9) 35.9 (0.4) 22.9 (4.3) 17.3 (0.3) 16.7 (3.4) 13.5 (0.2) 11.9 (1.0) 36.6 (1.7) 20.6 (0.3) 16.8 (0.5) 13.7 (0.7) 13.3 (0.7) 9.1 (0.3) 5.3 (0.1)
18:1(n-7) 5.7 (0.2) 11.1 (0.9) 12.6 (0.1) 15.3 (3.3) 13.4 (0.2) 12.5 (0.3) 2.3 (0.2) 9.7 (0.3) 10.6 (0.2) 11.6 (0.4) 10.4 (0.2) 14.1 (0.3) 17.7 (0.4)
18:2(n-6) 1.7 (0.2) 8.7 (1.2) 11.0 (0.1) 9.3 (3.8) 13.5 (0.4) 13.4 (0.4) 4.2 (0.1) 13.0 (0.1) 13.6 (0.1) 13.5 (0.6) 11.9 (0.3) 14.7 (0.3) 20.8 (0.3)
18:3 0 1.0 (0.2) 1.4 (0.1) 1.2 (0.7) 1.5 (0.1) 1.0 (0.1) 0 1.1 (0.1) 1.0 (0.1) 0.9 (0.2) 0.7 (0.1) 0.7 (0.1) 2.7 (0.1)
20:2(n-6) 0 1.3 (0.1) 1.8 (0.1) 1.7 (0.7) 2.1 (0.1) 1.7 (0.5) 0 1.6 (0.1) 1.9 (0.1) 2.1 (0.1) 1.6 (0.1) 2.7 (0.1) 4.7 (0.1)
20:3(n-6) 0 1.9 (0.3) 2.5 (0.1) 1.7 (1.3) 2.6 (0.1) 2.3 (0.2) 0 1.4 (0.2) 1.7 (0.3) 1.5 (0.1) 1.0 (0.1) 1.4 (0.1) 5.5 (0.2)
20:4(n-6) 6.0 (1.9) 8.2 (0.4) 10.3 (0.3) 7.2 (6.7) 11.7 (0.3) 15.3 (0.5) 7.9 (0.7) 12.3 (1.1) 14.9 (0.4) 16.5 (0.9) 18.3 (0.3) 18.8 (0.5) 8.4 (0.4)
20:5(n-3) 4.2 (0.3) 4.2 (0.5) 5.8 (0.2) 3.4 (3.4) 6.3 (0.3) 8.1 (0.1) 2.0 (0.3) 3.0 (0.2) 3.3 (0.1) 3.2 (0.3) 3.0 (0.2) 3.5 (0.1) 4.5 (0.2)
Total C20
PUFAs
10.2 (1.7) 15.7 (1.8) 20.5 (0.5) 14.1 (11.9) 22.7 (0.7) 27.4 (0.5) 10.1 (1.0) 18.3 (1.6) 21.8 (0.8) 23.3 (1.4) 24.0 (0.6) 26.4 (0.6) 24.2 (0.8)
Total no. of
fatty acids
19 28 28 28 28 28 15 23 24 24 24 24 28
Sat/unsat
ratioa
0.43 (0.02) 0.36 (0.02) 0.37 (0.01) 0.52 (0.20) 0.38 (0.02) 0.34 (0.02) 0.53 (0.03) 0.37 (0.03) 0.38 (0.02) 0.42 (0.03) 0.50 (0.03) 0.40 (0.01) 0.32 (0.01)
Odd/even
ratiob
0.025
(0.002)
0.051
(0.006)
0.070
(0.002)
0.110
(0.032)
0.089
(0.004)
0.104
(0.004)
0.01
(0.01)
0.04
(0.01)
0.05
(0.01)
0.07
(0.01)
0.10
(0.01)
0.12
(0.01)
0.17
(0.01)
Fatty acid compositions are mean percentages of total FAs (SD) and nZ3. All components present at O2% abundance during the experiment are shown.a Ratio of saturated-to-unsaturated fatty acids.b Ratio of odd-to-even carbon number fatty acids.
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Table 3
Fatty acids compositions of F. candida and P. minuta fed Z. mays for 39 days from day 0 of the diet-switching experiment
Fatty acids Time (days) Diet
F. candida P. minuta
0 2 4 8 16 39 0 2 4 8 16 39
16:0 12.5 (0.5) 18.9 (1.1) 18.0 (0.1) 18.0 (0.4) 11.9 (1.0) 12.4 (1.2) 15.1 (0.6) 17.8 (1.0) 18.3 (2.0) 17.7 (1.0) 18.9 (1.6) 26.7 (2.2) 22.6 (0.2)
16:1(n-7) 15.6 (1.7) 7.1 (0.8) 4.7 (0.5) 3.3 (0.3) 2.7 (1.1) 2.3 (0.7) 11.1 (2.1) 12.3 (2.1) 8.5 (1.7) 6.3 (0.6) 4.3 (1.6) 1.2 (0.1) 0.8 (0.1)
18:0 13.2 (1.0) 13.2 (0.3) 13.3 (0.2) 13.2 (0.1) 11.6 (1.9) 15.0 (1.2) 18.2 (1.9) 17.9 (2.0) 21.2 (2.9) 19.0 (2.5) 27.8 (4.0) 49.3 (3.5) 2.8 (0.3)
18:1(n-9) 35.9 (0.4) 34.1 (0.7) 33.1 (0.8) 34.9 (1.5) 31.4 (2.3) 39.8 (0.9) 37.7 (1.8) 36.5 (3.2) 35.6 (1.8) 32.9 (1.3) 28.6 (5.2) 15.4 (4.0) 20.4 (0.5)
18:1(n-7) 5.7 (0.2) 3.3 (0.3) 2.6 (0.2) 2.6 (0.1) 2.4 (0.7) 2.6 (0.5) 2.4 (0.2) 3.9 (0.4) 4.5 (0.3) 3.3 (0.1) 2.8 (0.1) 2.0 (0.4) 0.9 (0.1)
18:2(n-6) 1.7 (0.2) 10.5 (2.3) 10.8 (1.4) 11.2 (1.4) 18.2 (8.1) 8.3 (1.0) 4.3 (0.1) 3.7 (0.6) 4.1 (1.6) 6.5 (1.2) 6.6 (1.0) 0 43.1 (0.7)
18:3 0 1.2 (0.1) 1.7 (0.2) 1.5 (0.2) 2.1 (0.7) 0.9 (0.5) 0.6 (0.1) 0 2.5 (0.5) 0 0 0 5.5 (0.2)
20:4(n-6) 6.0 (1.9) 4.3 (0.7) 6.8 (0.1) 6.2 (1.7) 8.6 (0.3) 8.0 (1.4) 8.2 (0.7) 0.9 (1.6) 1.6 (1.4) 6.0 (2.9) 1.3 (2.2) 0 0
20:5(n-3) 4.2 (0.3) 1.9 (0.4) 3.4 (0.1) 3.2 (0.3) 5.2 (1.1) 3.8 (0.3) 2.1 (0.4) 0.2 (0.3) 0.1 (0.2) 1.5 (1.0) 0.3 (0.5) 0 0
Total C20
PUFAs
10.2 (1.7) 6.9 (1.1) 11.1 (0.1) 10.3 (0.9) 10.0 (8.7) 12.4 (1.8) 10.1 (1.0) 1.1 (1.9) 1.8 (1.5) 7.4 (3.9) 1.6 (2.7) 0 0
Total no. of
fatty acids
19 20 19 19 19 19 15 18 17 17 18 10 12
Sat/unsat
ratioa
0.42 (0.02) 0.55 (0.02) 0.53 (0.01) 0.53 (0.01) 0.36 (0.07) 0.47 (0.07) 0.53 (0.03) 0.63 (0.01) 0.74 (0.15) 0.67 (0.1) 1.05 (0.28) 4.23 (1.33) 0.41 (0.02)
Odd/even
ratiob
0.025
(0.002)
0.018
(0.002)
0.020
(0.001)
0.020
(0.004)
0.027
(0.001)
0.043
(0.018)
0.015
(0.002)
0.034
(0.003)
0.041
(0.010)
0.036
(0.008)
0.046
(0.009)
0.039
(0.003)
0.023
(0.012)
Fatty acid compositions are mean percentages of total FAs (SD) and nZ3. All components present at O2% abundance during the experiment are shown.a Ratio of saturated-to-unsaturated fatty acids.b Ratio of odd-to-even carbon number fatty acids.
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16
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Table 4
Fatty acids compositions of F. candida and P. minuta fed A. glutinosa for 39 days from day 0 of the diet-switching experiment
Fatty acids Time (days) Diet
F. candida P. minuta
0 2 4 8 16 39 0 2 4 8 16 39
14:0 1.3 (0.1) 1.4 (0.2) 1.3 (0.3) 1.4 (0.3) 1.2 (0.1) 0.6 (0.2) 1.8 (0.2) 1.3 (0.3) 1.5 (1.0) 1.5 (0.1) 1.0 (1.2) 0 2.6 (0.2)
15:0 0.3 (0.1) 0.4 (0.1) 0.6 (0.1) 1.1 (0.4) 1.2 (0.1) 0.6 (0.1) 0.8 (0.2) 0.9 (0.2) 1.0 (0.1) 1.9 (0.9) 0.8 (0.4) 0 5.4 (1.5)
16:0 13.9 (1.0) 14.4 (1.4) 14.4 (1.3) 13.8 (1.0) 10.5 (0.5) 10.0 (0.4) 27.2 (0.4) 24.8 (2.6) 24.4 (2.1) 25.2 (0.6) 18.0 (2.1) 10.2 (1.6) 26.6 (0.4)
16:1(n-7) 15.6 (0.4) 16.9 (0.6) 14.7 (0.2) 12.5 (1.7) 8.5 (1.2) 6.0 (2.7) 8.4 (4.6) 7.0 (1.0) 5.6 (0.9) 3.9 (0.6) 1.8 (1.0) Trace 1.3 (0.1)
16:2 Trace 0.3 (0.1) 0.5 (0.1) 1.1 (0.2) 1.0 (0.3) 0.8 (0.3) 0 0.8 (0.5) 1.1 (0.2) 1.5 (0.1) 2.8 (0.2) 0 0.6 (0.1)
17:0 0.3 (0.1) 0.5 (0.1) 0.7 (0.1) 1.1 (0.2) 1.2 (0.1) 0.9 (0.4) 1.6 (0.1) 2.2 (1.2) 4.8 (0.9) 4.7 (0.4) 3.1 (0.2) 0 0.5 (0.1)
18:0 11.2 (1.2) 12.7 (1.1) 16.0 (0.6) 17.3 (1.6) 14.6 (0.5) 20.2 (4.0) 29.8 (2.3) 30.3 (1.9) 26.8 (3.0) 27.0 (1.6) 31.8 (6.2) 30.6 (5.5) 6.0 (0.1)
18:1(n-9) 38.0 (0.3) 38.6 (0.8) 38.9 (1.8) 36.8 (1.9) 32.9 (0.9) 33.4 (1.4) 16.5 (2.2) 24.6 (2.8) 23.2 (1.0) 19.9 (2.0) 17.4 (1.0) 13.0 (1.4) 9.4 (0.2)
18:1(n-7) 5.6 (0.1) 6.6 (0.4) 7.2 (0.4) 7.6 (0.3) 6.5 (0.7) 3.6 (2.5) 5.2 (4.8) 4.0 (0.7) 5.0 (0.3) 5.7 (0.5) 6.1 (1.0) 4.3 (0.5) 1.7 (0.1)
18:2(n-6) 1.7 (0.1) 1.3 (0.7) 0.7 (0.2) 0.7 (0.1) 1.9 (0.1) 1.4 (0.9) 1.8 (0.8) 0 0 0 4.0 (0.7) 3.0 (0.4) 9.7 (0.2)
18:3 0.5 (0.1) 0.3 (0.1) 0.3 (0.1) 0.6 (0.1) 0.9 (0.1) 0.6 (0.1) 0 0 0 0 0 1.5 (0.4) 6.8 (0.3)
20:0 0.3 (0.1) 0.4 (0.1) 0.7 (0.1) 1.2 (0.4) 0.8 (0.1) 1.8 (2.3) 0 0 0 0 2.2 (0.8) 4.2 (1.0) 9.1 (0.3)
20:1 0.6 (0.1) 0.7 (0.1) 1.3 (0.1) 1.7 (0.2) 1.4 (0.2) 3.6 (3.6) 1.4 (0.5) 0 0 0 3.4 (0.1) 18.3 (2.5) 0
20:4(n-6) 5.1 (0.3) 2.7 (2.2) 0 0 8.2 (2.1) 11.3 (2.8) 0 0 0 0 0 7.1 (0.4) 0
20:5(n-3) 2.9 (0.2) 1.3 (1.4) 0 0 6.0 (0.4) 7.0 (1.6) 0 0 0 0 0 0 0
22:0 0 0 0 0 0 0 0 0 0 0 0 0 12.6 (0.2)
24:0 0 0 0 0 0 0 0 0 0 0 0 0 4.8 (1.0)
Total C20
PUFAs
8.1 (0.1) 4.0 (3.6) 0.5 (0.5) 0 14.2 (2.4) 17.8 (3.6) 0 0 0 0 0 0 0
Total no. of
fatty acids
20 19 19 17 19 20 16 13 15 15 17 12 18
Sat/unsat
ratioa
0.40 (0.01) 0.45 (0.06) 0.54 (0.05) 0.63 (0.09) 0.48 (0.01) 0.56 (0.17) 1.05 (0.28) 1.53 (0.18) 1.48 (0.12) 1.78 (0.12) 1.73 (0.18) 0.89 (0.22) 2.32 (0.05)
Odd/even
ratiob
0.016
(0.001)
0.021
(0.002)
0.036
(0.004)
0.056
(0.013)
0.046
(0.001)
0.069
(0.072)
0.512
(0.130)
0.052
(0.013)
0.128
(0.011)
0.164
(0.004)
0.144
(0.026)
0.047
(0.014)
0.083
(0.015)
Fatty acid compositions are mean percentages of total FAs (SD) and nZ3. All components present at O2% abundance during the experiment are shown.a Ratio of saturated-to-unsaturated fatty acids.b Ratio of odd-to-even carbon number fatty acids.
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P.M. Chamberlain et al. / Soil Biology & Biochemistry 37 (2005) 1608–1624 1615
than the other diets (P!0.005). The ratios of odd-to-even
chain length FAs of the diets were also significantly
different between diets (P!0.005); post-hoc comparisons
showed that P. redivivus and alder possessed significantly
higher ratios of odd-to-even FAs than maize and
C. cladosporioides, and that the ratios of odd-to-even FAs
of P. redivivus and alder were also significantly different to
each other.
3.2. Collembolan fatty acid compositions
3.2.1. Collembola switched to C. cladosporioides
Relative to day 0, a wider range of FAs was detected in
F. candida fed C. cladosporioides on all other days, despite
the narrower range of FAs present in the diet (Table 1).
However, these additional components were present only at
low relative abundances. The abundance of 16:1(n-7) in the
FA pool fell significantly (P!0.001) during the experiment,
as did the abundance of 18:1(n-7) (P!0.01); the reduced
abundances of these FAs reflected their low abundance
in the C. cladosporioides diet. Conversely, 18:2(n-6)
increased in abundance significantly (P!0.001) during
the experiment, reflecting the high abundance of this FA in
the diet. The 18:0 component was maintained at higher
proportions of total FAs in F. candida compared to the diet
while 16:0 and 18:1(n-9) remained at abundances similar to
those of the diet throughout the experiment. The PUFAs
20:4(n-6) and 20:5(n-3) were observed sporadically during
the experiment but were not detected in C. cladosporioides.
There were no significant differences in the ratio of
saturated-to-unsaturated FAs of F. candida during the
experiment, which remained similar to the saturated-to-
unsaturated FA ratio of the diet. Similarly, the ratio of odd-
to-even chain length FAs did not change significantly during
the experiment, and remained higher than that of the diet.
The range of FAs observed in P. minuta fed
C. cladosporioides was less than that observed in
F. candida. In P. minuta, 18:0 was maintained at higher
proportions of total FAs relative to the diet, while 16:0 and
18:1(n-9) remained relatively constant in abundances
similar to the diet. As in F. candida, 16:1(n-7) fell
significantly (P!0.005) in relative abundance over time
while 18:2(n-6) increased significantly (P!0.005) from
4.5% on day 0 to 10.3% on day 4 but remained around 3%
for the remainder of the experiment. The PUFAs 20:4(n-6)
and 20:5(n-3) were observed in P. minuta on days 0–4 only.
There were no significant changes in the ratios of saturated-
to-unsaturated and odd-to-even FAs in P. minuta.
3.2.2. Collembola switched to P. redivivus
A greater range of FAs was observed in Collembola fed
P. redivivus than for any other diet, reflecting the wide range
of FAs in the nematode diet (Table 2). Significant (all P!0.01) increases in abundance were observed in i15:0, i17:0,
18:1(n-7), 18:2(n-6) and 18:3. All these components were
present in the dietary FA composition at relatively high
abundances. However, significant decreases were observed
in some components present in F. candida in high
abundances on day 0, particularly 16:0 (P!0.001),
16:1(n-7) (P!0.001) and 18:1(n-9) (P!0.001), reflecting
the lower abundance of these components in the diet relative
to their abundance in F. candida on day 0. Increases in the
concentrations of C20 PUFAs were also observed in
F. candida; on day 0 PUFAs accounted for 10.7% of the
total FA composition but by day 39 they amounted to 27.4%
of the total FA complement. However, these differences
were not significant. There were no significant differences in
the ratio of saturated-to-unsaturated FAs in F. candida over
time and the saturated-to-unsaturated FA ratio of the diet
was not significantly different to the ratio of saturated-to-
unsaturated FAs in F. candida (t-test, P!0.17) on day 39.
The ratio of odd-to-even chain length FAs increased
significantly (P!0.005) from 0.02 on day 0 to 0.1 on day
39 but the ratio of odd-to-even FAs in the diet was still
significantly greater (t-test, P!0.005) than that of
F. candida on day 39.
Similar effects were observed in P. minuta fed
P. redivivus. The FAs i15:0, i17:0, 18:1(n-7) and
18:2(n-6) all rose in abundance significantly (P!0.005)
during the experiment. Conversely, the abundances of 16:0,
16:1(n-7) and 18:1(n-9) fell significantly (P!0.005) during
the experiment. The proportion of C20 PUFAs in the FA
pool increased from 10.1% on day 0 to 28.5% on day 39.
There were no significant changes in the ratio of saturated-
to-unsaturated FAs in P. minuta fed nematodes. However,
the ratio of odd-to-even chain length FAs increased
significantly (P!0.005) from 0.01 to 0.12 during the
experiment; the ratio on day 39 was significantly lower (t-
test, P!0.05) than that of the diet. All changes in
Collembola FA compositions reflected the abundance of
the corresponding dietary components.
3.2.3. Collembola switched to Z. mays
The total number of FAs detected in F. candida fed
maize did not change during the experiment although the
relative abundances of some FAs did change significantly.
The abundance of 18:2(n-6) changed significantly (P!0.001) from 1.7% on day 0 to 18.2% on day 16 before
falling to 8.3% on day 39, reflecting the high abundance of
18:2(n-6) in maize. Conversely, the significant reductions in
the proportions of 16:1(n-7) (P!0.005) reflected the low
abundance of this component in the diet. The proportions of
16:0 in F. candida were generally lower than those of 16:0
in the diet, while the abundances of 18:0 and 18:1(n-9) were
higher than those of the corresponding dietary components.
The abundances of the PUFAs 20:4(n-6) and 20:5(n-3) were
relatively constant throughout the experiment despite their
absence from the diet. There were no significant changes in
the ratio of saturated-to-unsaturated and odd-to-even FAs in
F. candida. Both the ratios of saturated-to-unsaturated and
odd-to-even FAs of the collembola were similar to those of
the maize diet.
P.M. Chamberlain et al. / Soil Biology & Biochemistry 37 (2005) 1608–16241616
Post-hoc comparisons of the FA compositions of
P. minuta fed maize demonstrated that samples taken on
day 39 were significantly different (P!0.005) to those on
all other days. On day 39, almost half the FA composition
was attributable to one component (18:0) and no PUFAs
were detected. This led to the extremely high ratio of
saturated-to-unsaturated FAs observed which was signifi-
cantly different (P!0.005) to those observed on all other
sampling days. Excluding day 39, the abundance of 16:0 in
P. minuta was maintained at proportions similar to that of
the diet while 18:0 and 18:1(n-9) were maintained at higher
abundances in the Collembola relative to those of the diet.
During the experiment, the abundance of 16:1(n-7) fell
significantly (P!0.005) from 11.1 to 1.2%. However, the
abundance of 18:2(n-6) in P. minuta did not increase over
time despite the high abundance of this component in the
diet. There were no significant changes in the ratio of odd-
to-even chain length FAs of P. minuta, and the ratio on day
39 was not significantly different to that of the diet (t-test,
P!0.05).
3.2.4. Collembola switched to A. glutinosa
The range of FAs observed in F. candida fed alder
changed little during the diet-switch experiment, although
the abundances of the some FAs did change significantly
over time. Significant decreases in the abundance of
16:1(n-7) (P!0.005) were observed. The 18:0 and
18:1(n-9) components of F. candida were maintained at
higher proportions than the same components in the diet,
while 16:0 was consistently lower in abundance in F.
candida than the corresponding dietary FA. The PUFAs
20:4(n-6) and 20:5(n-3) were observed in F. candida at the
beginning and end of the experiment, despite their absence
from the diet, while the long chain FAs 22:0 and 24:0 were
not detected in F. candida despite their presence in the diet
in relatively high abundances. The ratio of saturated-to-
unsaturated FAs of F. candida did not change significantly
with time and was consistently lower than that of the diet.
There were also no significant differences over time in the
ratio of odd-to-even chain length FAs in F. candida
consuming alder.
The FAs of P. minuta fed alder were qualitatively
different on day 39 to those on all other days. In particular,
many of the components present at low abundances on days
0–16 were entirely absent on day 39 and the 20:1 component
exhibited a high abundance of 18.3% which was
Table 5
Total abundances of C20 PUFAs in F. candida and P. minuta fed the four diets f
C. cladosporioides P. redivivus
F. candida 4.48cq 18.83a
p
P. minuta 1.77eq 21.31d
p
Data are means of percent total FAs. Superscripts refer to comparisons within C
letters represent significant differences (ANOVA; P!0.05).
significantly higher than on all other days (P!0.005).
During the experiment the 16:1(n-7) component fell in
abundance significantly (P!0.001), from 8.4% on day 0 to
only trace levels on day 39, while 16:0 fell in abundance
significantly in the latter part of experiment (P!0.005).
Consistently higher proportions of the 18:0 and 18:1(n-9)
components were found in P. minuta relative to the alder
diet. The PUFA 20:4(n-6) was only detected on day 39
while 20:5(n-3) was not detected at all in P. minuta fed
alder. There were no significant differences over time in the
ratio of saturated-to-unsaturated FAs of P. minuta,
although all ratios were lower than that of the saturated-
to-unsaturated FA ratios of the diet. There were significant
differences over time in the ratio of the odd-to-even
chain length FAs of P. minuta (P!0.05); post-hoc
comparisons showed that the ratio on day 0 was signifi-
cantly higher (P!0.05) than for all other days.
3.2.5. Occurrence of C20 PUFAs in Collembola
The four diets significantly affected the abundance of C20
PUFAs in both Collembolan species (Table 5; P!0.05);
post-hoc comparisons demonstrate that the P. redivivus diet
resulted in a significantly higher C20 PUFA content than all
other diets. In F. candida, feeding on C. cladosporioides and
alder resulted in the lowest PUFA contents, while the maize
diet resulted in intermediate abundances of the PUFAs. In
P. minuta, abundances of PUFAs when fed C. cladospor-
ioides, maize and alder were low and not significantly
different among the diets. However, P. minuta fed
P. redivivus displayed a 10-fold increase in the abundance
of C20 PUFAs relative to P. minuta fed the other diets.
Regardless of the diet, P. minuta exhibited significantly
higher ratios of saturated-to-unsaturated (P!0.005) and
odd-to-even (P!0.005) FAs relative to F. candida.
3.2.6. Rate of change of FA compositions
The relative abundance of many Collembolan FAs
changed during the diet-switch experiment. Many of these
changes were exhibited as a gradual increase or decrease
over time or fluctuations about a mean value. However for
some FAs the rate of change of abundance could be
quantified by fitting negative exponential equations to the
data. For example, in all diet-switches the abundances of
16:1(n-7) decreased over time (Fig. 2) and could be fitted to
such equations. Half-lives of the 16:1(n-7) abundance
changes varied between 1.4 and 13.1 days, and ANOVA
or 39 days
Z. mays A. glutinosa
9.49br 5.81c
t
2.22es 1.33e
u
ollembolan species, subscripts refer to comparisons within diets. Different
Fig. 2. Changes in the relative concentrations of 16:1 (n-7) in all diet-switch experiments. Data are percentages of total fatty acids GSD (nZ3).
P.M. Chamberlain et al. / Soil Biology & Biochemistry 37 (2005) 1608–1624 1617
of the rate coefficient in the equations demonstrated that the
rates of change of this FA in F. candida were significantly
affected by the diets (P!0.005). However, the rates of
change of 16:1 (n-7) in P. minuta were not significantly
different between diets (P!0.3). In the nematode diet-
switches, the changes in abundance of most major FAs, with
the exception of 18:0, could be fitted to negative exponential
equations (Figs. 3 and 4). The half-lives of FA abundance
changes varied between 1.2 and 22.4 days in F. candida, and
0.5 and 7.7 days in P. minuta, and the rate of change of
Collembolan FA abundance was not related to the FA
composition of the diet (R2Z0.27, P!0.3). The rates of
change of abundance of i15:0, 16:0, i17:0 and 20:4 (n-6)
were all significantly different (Tukey, PO0.05) between
the two species, but the species exhibiting the greater rate of
change varied between FAs.
3.2.7. PCA of Collembolan fatty acid compositions
The FAs of the Collembola sampled on day 39 (the final
day) of the experiment were subjected to principle
components analysis (Figs. 5 and 6). The FAs derived
from both species of Collembola fed the differing diets
clustered separately using the PCA scores, indicating that
the effects of each diet on FA compositions was different
and consistent across all replicates. The first three axes
(Fig. 5) accounted for 36.0, 20.1 and 15.0%, respectively, of
the variation in the FA compositions and therefore together
accounted for 71.1% of the total variance. PC1 was mainly
described by the FAs which exhibited high abundances in
Collembola fed P. redivivus (Fig. 6) with negative
contributions from the n-alkanoic FA components. PC2
was mainly described by components that exhibited less
than 5% of the FA composition on day 39, i.e. 18:1(n-5),
20:0 and 20:1, with negative contributions from 16:1(n-7).
4. Discussion
4.1. Dietary FA compositions
The four diets represent a broad range of FA compo-
sitions. In total, 33 different FAs were detected in the diets,
although only 9 FAs were common to all; the abundances of
16:0, 18:1(n-9), 18:1(n-7), 18:2(n-6) and the C20 PUFAs
were particularly variable between diets. In our study, a total
of 30 FAs were detected in P. redivivus while other soil
nematodes have been reported to contain 13–22 individual
FAs (Sivapalan and Jenkins, 1966; Holz et al., 1999; Ruess
et al., 2002). For earlier work especially, this increase in
diversity of FAs is most likely due to the improved detection
limits and resolution facilitated by modern gas chromato-
graphic techniques. Sivapalan and Jenkins (1966) reported
that the FA compositions of P. redivivus was dominated by
18:1 and 18:2 in abundances (16.3 and 20.7%, respectively)
similar to those reported here. C20 PUFAs, especially
20:4(n-6) and 20:5(n-3), are often major components of
nematode FAs (Tanaka et al., 1996; Chen et al., 2001).
However, in contrast to other studies on P. redivivus
(Sivapalan and Jenkins, 1966), no FAs were observed
eluting after 20:5(n-3) in our work. The C20 PUFAs were
not present in the fungal, maize and alder diets. The
observed FA compositions of maize is similar to that
reported by Woodbury et al. (1998), but as far as can be
ascertained, there have been no reports of the FA
Fig. 3. Changes in the relative concentrations of the major fatty acids of F. candida fed a diet of P. redivivus. Data are percentages of total fatty acids GSD
(nZ3).
P.M. Chamberlain et al. / Soil Biology & Biochemistry 37 (2005) 1608–16241618
compositions of A. glutinosa or C. cladosporioides. The
lipid content of laboratory-grown fungi is dependent upon
the culture conditions (Griffiths et al., 2003) and since C.
cladosporioides was cultured on a matrix consisting of only
one C source, sucrose, all FAs present in the
C. cladosporioides must be the result of fungal biosynthesis.
The wide range of FAs present in these diets demonstrates
that Collembola in the soil are likely to be exposed to a wide
variety of FAs as they forage.
4.2. Collembolan FA compositions
The FA compositions of Collembola are a little-studied
area. Stransky et al. (1986) identified 29 FAs in the field-
derived specimens of Tetrodontophora bielanensis while
Holmstrup et al. (2002) and Ruess et al. (2004) reported 14
and 11–21 FAs in various Collembolan species, respectively.
In our work, 13–28 FAs were detected in F. candida
and P. minuta with the range of components depending upon
diet; this was similar to the previous reports. Holmstrup et al.
(2002) investigated the phospholipid and neutral lipid FAs of
F. candida under varying moisture regimes and found a shift
towards greater unsaturation in the PLFAs of drought-
acclimatised individuals but little change in the NLFAs.
Since the neutral fraction comprised 89% of the total FAs, the
change in the total FAs content upon droughting was small.
Similarly, Haubert et al. (2004) starved the Collembolan
Protaphorura fimata and established that whilst the NLFA
content of the Collembola fell the NLFAs were equally
metabolised so the overall FA composition remained
relatively unchanged. The NLFAs represent the major FA
pool in Collembola; even in P. fimata starved for 28 days the
NLFAs accounted for ca. 90% of the total FAs. We have
previously reported the total FA compositions of F. candida
and P. minuta fed continuously on a diet of bakers yeast
(Chamberlain et al., 2004) and demonstrated that
Fig. 4. Changes in the relative concentrations of the major fatty acids of P. minuta fed a diet of P. redivivus. Data are percentages of total fatty acids GSD
(nZ3).
P.M. Chamberlain et al. / Soil Biology & Biochemistry 37 (2005) 1608–1624 1619
the composition did not change significantly with time.
However, changing the diet in the current work caused
significant changes in the FA compositions of both
Collembolan species as observed for F. candida, P. fimata
and Heteromurus nitidus fed a range fungal and nematode
diets (Ruess et al., 2004). Some components present in low
abundance in Collembola, such as i15:0 and i17:0, are
characteristic of bacterial FAs and are likely derived from gut
bacteria (Jantzen and Bryn, 1985). However, all other
components are derived from Collembolan lipids since the
guts were empty of food when FAs were extracted.
Fatty acids in invertebrates can either be directly
assimilated from the diet or biosynthesised by the consumer
(Canavoso et al., 2001) although it is energetically
favourable to directly incorporate dietary lipid and catabo-
lise dietary carbohydrate (Pond, 1981). Therefore, if
changes in individual FA abundances can be directly linked
to the abundance of FAs in the diet, there is a strong case for
the direct assimilation of these dietary components. For
example, in our work the abundances of 16:1(n-7) in each of
the four offered diets were under 2% and since there was a
gradual reduction of 16:1(n-7) in Collembola in all diet
switches, it is probable that the Collembola did not
biosynthesise significant proportions of this component.
The high abundance of this FA in the diet on day 0 is likely
due to the high abundance in the initial bakers yeast diet
(32–37%; Chamberlain et al., 2004) again consistent with
direct assimilation from the diet. Similar trends were
observed in 18:1(n-7) in F. candida fed C. cladosporioides
and Z. mays, where the abundance of this component fell in
response to low abundances in the diet. The opposite trend
was observed in F. candida fed nematodes where the
proportion of 18:1(n-7) more than doubled during the
experiment. This was consistent with a high input of this
Fig. 5. Principle components analysis showing variation in the fatty acid compositions of F. candida and P. minuta fed the four offered diets after 39 days of
consumption.
P.M. Chamberlain et al. / Soil Biology & Biochemistry 37 (2005) 1608–16241620
component from the diet. The components 16:1(n-7) and
18:1(n-7), along with other components of the nematode
diet which were incorporated into the Collembola, such as
i15:0 and i17:0, therefore appear to be FAs the Collembola
are capable of assimilating if available but do not
biosynthesise in high abundance if absent from the diet.
However, i15:0 and i17:0 are also present in gut bacteria,
Fig. 6. Individual fatty acid loading plot of the first two eigenva
and the identification of low abundances of these com-
ponents in Collembolan extracts is more likely due to these
bacteria than to nematode consumption. However, high
abundances of these FAs should be indicative of nematode
predation. Ruess et al. (2004) also identified 20:1(n-7) and
20:1(n-9) as biomarkers of nematode consumption since
these FAs were only found in Collembola fed these diets.
lues (PC1 and PC2) of the principle components analysis.
P.M. Chamberlain et al. / Soil Biology & Biochemistry 37 (2005) 1608–1624 1621
However, 20:1 is also present in F. candida and P. minuta
feeding on alder, probably through desaturation of the large
abundances of dietary 20:0. Caution is therefore advisable
in using these FAs, present in only low relative concen-
trations in Collembola, as biomarkers of specific diets until
further studies have clarified the factors causing their
presence in Collembolan FAs.
The FA 18:2(n-6) is a biochemical precursor of 18:3(n-6)
and 20:4(n-6) and is an essential FA for many, but not all,
invertebrates (Blomquist et al., 1991; Weinert et al., 1993).
The presence of 18:2(n-6) on day 0, albeit at low
abundances, is evidence for the biosynthesis of this FA in
Collembola, since it was not present in the initial yeast diet
(Chamberlain et al., 2004). High abundances of 18:2(n-6) in
the C. cladosporioides, P. redivivus and maize diets caused
significant increases in the abundance of this FA in
F. candida consistent with the direct incorporation of this
component. However, the relatively high abundance of
18:2(n-6) in alder did not cause an increase in the proportion
of this component in F. candida, demonstrating that even if
a particular FA is present in the diet it may not be directly
incorporated to abundances similar to those of the diet. This
was also the case in P. minuta, where high dietary
abundance of 18:2(n-6) in P. redivivus resulted in increased
abundances in the Collembola; high dietary proportions in
the other three diets did not consistently produce high
concentrations in the consumers.
Direct incorporation of FAs is also likely to contribute to
those Collembolan FAs that did not change in abundance
during the diet-switch experiments. In such components, the
concentration of a particular FA would be the result of a
combination of direct incorporation from the diet and
biosynthesis either from acetate or via modification of other
FAs (Canavoso et al., 2001). In our experiments, dietary
abundances of 18:0 and 18:1(n-9) were generally lower than
those of the Collembola, consistent with biosynthesis and
resulting in increased abundances of these FAs within the
Collembola. Both F. candida and P. minuta fed maize
biosynthesised high proportions of 18:0 likely from dietary
18:1(n-9) and 18:2(n-6). However, P. minuta maintained
higher proportions of 18:0 in tissue FAs than F. candida. It
is possible that P. minuta prefers to store FAs as C18
unsaturated and monounsaturated components rather than
C18 PUFAs since 18:2(n-6) has a specialised function as a
C20 PUFA precursor although the precise reasons for these
observations remains unclear. The Collembolan 16:0
component generally exhibited abundances equal to or
lower than those of the diet such that the diet could have
supplied all the 16:0 observed in the Collembola with no
biosynthetic contribution from the consumers. In the
nematode diet switch, the abundances of 16:0 in Collembola
decreased over time in response to low dietary abundances
indicating that the Collembola, when fed nematodes, did not
biosynthesise high amounts of 16:0. This strongly contrasts
with 18:0 and 18:1(n-9) in Collembola fed nematodes which
were maintained at abundances greater than those of the diet
throughout the experiment.
4.3. C20 PUFAs in Collembola
Collembola have been shown to be more closely related
to crustacea than to insects (Nardi et al., 2003). The
occurrence of high abundances of C20 PUFAs in Collem-
bola, already reported by Holmstrup et al. (2002) and
Chamberlain et al. (2004) and expanded here for a range of
different diets, is further evidence for the differentiation of
Collembola and insects since terrestrial insects generally do
not biosynthesise such high abundances of PUFAs (Stan-
ley-Samuelson et al., 1988). Crustacea, however, often
contain very high abundances of PUFAs (Graeve et al.,
2001). Our results indicate that Collembola biosynthesise
high abundances of 20:4(n-6) and 20:5(n-3) and that
Collembolan PUFA abundances may be affected by those
of the diet. However, Ruess et al. (2004) reported no
20:5(n-3) in F. candida, H. nitidus and P. fimata but low
abundances of 20:2(n-6) and 20:3(n-6) in some individuals.
Therefore, 20:5(n-3) may not always be present when C20
PUFAs are detected. Not surprisingly, of the four offered
diets the nematode diet produced the highest abundances of
C20 PUFAs in Collembola since 24% of the FA profile of
nematodes was comprised of these components. However,
the remaining three diets, which did not contain C20 PUFAs,
all produced higher abundances of these components in F.
candida than in P. minuta. The reason for the lower
abundances of PUFAs in P. minuta is not clear although it is
likely to be related to differing metabolism in the two
species.
4.4. FA compositions of Collembola fed nematodes
The changes in the Collembolan FA composition in
response to the nematode diet were particularly striking.
The compositions of both species and the ratios of odd-to-
even carbon number FAs, grew to mirror those of the diet
during the experiment. Additionally, individual compounds
present in the nematode diet were observed in Collembolan
FAs at abundances similar to the diet. This reveals the
surprising plasticity of the FA profile of Collembola with a
nematode diet although the reason for the very different
response of the Collembola to this diet is not clear. We did
not explore the partitioning of the FAs into different
biochemical fractions but these observations suggest that
the substantial change in FA composition is due to direct
input of dietary FAs into NLFAs, rather than PLFAs, since
the functioning of PLFAs is closely related to their
molecular structure and hence PLFA content is tightly
regulated by the organism (Hazel and Williams, 1990;
Holmstrup et al., 2002). Ruess et al. (2004) also fed
nematodes to Collembola but did not observe the same
modifications to Collembolan FA compositions. This was
mainly due to the contrasting FA compositions of the
P.M. Chamberlain et al. / Soil Biology & Biochemistry 37 (2005) 1608–16241622
nematodes used which mostly did not contain high
abundances of i15:0, i17:0 and 18:1(n-7). Consumption of
the nematode species that did contain the latter two FAs
resulted in elevated proportions of these compounds in
Collembola NLFAs, consistent with direct incorporation
from the diet. However, high abundances of 20:4(n-6) in
nematodes did not result in elevated abundances of this
component in the Collembola, unlike in our study. Further
work on other species that consume nematodes is therefore
necessary to establish whether these substantial shifts in the
FA composition are exhibited in other Collembola and
indeed in other invertebrates, consuming nematodes.
4.5. Rate of change of FA compositions
The significant changes in abundance observed in some
FAs (i.e. 16:1(n-7) in all switches and most major FAs in the
nematode switches (Figs. 2–4)) could be described using
negative exponential equations. This is not unexpected since
other researchers have demonstrated that the metabolism of
radiolabelled FFAs in insect haemolymph follows similar
negative exponential patterns (Downer and Chino, 1985;
Soulages and Wells, 1994; Atella et al., 2000). However,
whilst turnover of FFAs in haemolymph occurs in ca. 1 h,
turnover of FAs in diacylglycerol takes up to 10 h (Downer
and Chino, 1985). Since NLFAs are energy storage
molecules turnover times of these components would be
expected to be much longer still. Absolute turnover rates of
these FAs cannot be derived from the equations presented
since the FAs were not simply turning over; rather, they
were accumulating in, or being removed from, the
Collembolan lipid pool. However, the identification of
abundance changes that could be fitted to negative
exponential equations implies that these components were
being added to, or being removed from, the Collembolan
lipid pool strictly according to their presence in or absence
from the diet. This contrasts with, for example, 18:0 in
Collembola fed nematodes (Figs. 3 and 4); the abundances
of this component did not change significantly (Tukey, P!0.15) during the experiment, consistent with physiological
regulation of this FA by the Collembola.
Upon starvation, the absolute NLFA content of Collem-
bola is reduced rapidly over the first 14 days of starvation
(Haubert et al., 2004) and there is complete C turnover in
Collembolan FAs in ca. 30–40 days (Chamberlain et al.,
2004). The present results concur with those previously and
give some indication as to the period over which the FA
compositions of Collembola would be expected to change,
given a change in diet. In our study, half-lives varied
between 0.5 and 22.4 days demonstrating that, for
Collembola, the FA profile only represents the dietary
input and Collembolan biosynthesis of recent days and that
some components may appear and disappear, due to a
change in diet, at much greater rates than others. The factors
affecting the rates of change of FA abundances are as yet not
clear but are likely to include physiological differences
between the two species, and the lipid content of the diet.
4.6. Identification of FA trophic biomarkers
The changes in Collembolan FA compositions, as shown
by consideration of both individual FAs and PCA, indicate
that FA profiles do offer a means of elucidating trophic
preferences in Collembola although the FA content is
generally not simply related to that of the diet. In the PCA,
the main effect was the abundance of nematode-derived FAs
(i15:0, i17:0 and the PUFAs) which was not surprising given
the high input of dietary FAs directly into Collembolan
lipids when nematodes were consumed. Ruess et al. (2002)
concluded, also using PCA, that the FA compositions of
nematodes are affected by the FA content of their fungal
diets although no specific compounds were able to function
as biomarkers since all the various fungal diets did not
contain unique FAs. In our work, the offered diets contained
a much wider range of FAs and it would be possible, for
example, to use i15:0, i17:0 and 18:1(n-7) as biomarker
compounds for nematode predation since these compounds
only occurred at high abundance in the nematode diet and
when P. redivivus was consumed the abundance of these
particular FAs rose in the Collembola to abundances similar
to those of the diet. Components such as 20:4(n-6) and 20:5
(n-3), present at high abundance in the nematodes, could
not, however, be used as biomarkers for nematode predation
since it is clear from the present results that Collembola
biosynthesise these compounds in high abundance when not
present in the diet. The proportion of 18:2(n-6) could
potentially be used as a biomarker since it is evident that
Collembola biosynthesise only low abundance of this FA if
not present in the diet and that high abundances of this
component in Collembolan FAs are the result of incorpor-
ation from the diet. However, since not all high dietary
abundances of 18:2(n-6) result in high abundances in the
Collembola (e.g. in P. minuta, and F. candida when fed
alder) the impact of each diet on Collembolan FA profiles
must first be determined.
The high abundances of C20 PUFAs in Collembola also
has implications for the analysis of whole-soil FAs. Chen et
al. (2001) suggested that analyses of such components could
be useful as biomarkers for nematodes in soils as long as
insects and other soil dwelling invertebrates, which until
recently were thought to contain C20 PUFAs at low
abundances, could be removed from the samples prior to
extraction. The results presented here make this qualifica-
tion even more important since Collembola can contain
considerably higher abundances of C20 PUFAs than
previously realised. Even if only the PLFA fraction of soil
extracts is considered, Collembola could still represent a
significant contribution of FAs to this pool, since Holmstrup
et al. (2002) demonstrated that when fed bakers yeast, C20
PUFAs comprise 24% of the total PLFAs of F. candida.
Therefore careful removal of invertebrates such as
P.M. Chamberlain et al. / Soil Biology & Biochemistry 37 (2005) 1608–1624 1623
Collembola will be necessary before using PUFAs as
biomarkers for nematodes.
4.7. Conclusions
On the basis of the results presented here, we must re-
emphasise the conditions that must be fulfilled if
particular FAs are to be used as biomarkers in trophic
studies: (i) the FA compositions of the possible diets must
each contain unique compounds in relatively high
abundances, (ii) these compounds must be either absent
from, or only minor components of, those FAs biosynthe-
sised by the consumer, and (iii) these compounds must be
assimilated into the consumer FA composition as intact
molecules at significant abundance when the diet is
consumed. It is not sufficient to assume that if a particular
FA is present in a diet this compound will be assimilated
unchanged into the lipids of the consumer. These
considerations suggest that, prior to choice experiments,
FA components of a consumer fed on single diets must be
analysed in order to determine the FAs biosynthesised by
the organism and the effect of exogenous compounds on
FA compositions. Simply obtaining invertebrates from the
soil and analysing their FA composition, without a
thorough analysis of dietary FAs, is unlikely to yield
usable dietary information.
In summary, Collembola both directly assimilated from
the diet and biosynthesised FAs, although the pathways of
biosynthesis were not determined. Collembolan FA com-
positions were modified by all offered diets and the
nematode diet caused the largest changes. Biomarkers
for nematode consumption were identified, and the FA 18:2
(n-6) may also function in F. candida as a biomarker for
diets containing high abundances of this component
although not all diets rich in 18:2(n-6) produced elevated
proportions of this FA in this species. Our results indicate
that the influences of dietary FA compositions on Collem-
bolan FA compositions are complex and that a thorough
understanding of the impact of diet FAs on consumer FA
profiles is essential when using FAs as biomarkers in soil
food web studies.
Acknowledgements
This study was undertaken while PMC was in receipt
of a NERC CASE studentship. The use of the NERC
Mass Spectrometry facilities is gratefully acknowledged.
Margaret Hurley is thanked for statistical analysis and
advice.
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