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All content in this area was uploaded by Michał Filipiak on Nov 13, 2017
Content may be subject to copyright.
Physiological Entomology (2016), DOI: 10.1111/phen.12168
Nutritional dynamics during the development
of xylophagous beetles related to changes
in the stoichiometry of 11 elements
MICHAŁ FILIPIAK and JANUARY WEINER
Institute of Environmental Sciences, Faculty of Biology and Earth Sciences, Jagiellonian University, Kraków, Poland
Abstract. The present study examines the adaptive strategy used by wood-boring
beetles to compensate for the lack of nutrients in dead wood. The contents of nutritional
elements in growing wood-boring beetles (Stictoleptura rubra L. and Chalcophora
mariana Dejean) are compared with the elemental composition of decaying dead
wood (pine stumps), showing changes during the beetles’ ontogenetic (i.e. larval)
development. The stoichiometric ratios of C and other nutritional elements (N, P, K, Na,
Ca, Mg, Fe, Zn, Mn and Cu) are investigated to identify the most important nutrients
for larval development. The degree of nutritional mismatch that is encountered by
the beetle larvae changes dramatically over 3– 4 years of simultaneous larval growth
and wood decay. Excluding C, the relative contents of nutritional elements increase
substantially in decaying wood, whereas the opposite tendency is found in larvae, most
likely because of carbon deposition in fat. The elements limiting larval development
because of their scarcity in dead wood are N, P, K, Na, Mg, Zn and Cu. Fungal activity
(i.e. the transport of nutrients from the surrounding environment to decaying stumps)
can explain the observed mitigation of the original mismatch, although prolongation of
the larval development time is still necessary to compensate for the scarcity of some of
the required elements in food.
Key words. Beetle, deadwood, ecological stoichiometry, fungi, life history, wood
eater.
Introduction
Dead wood is one of the most abundant food resources in ter-
restrial ecosystems. However, its extreme nutritional deciency
poses great challenges for xylophages because it results in
severe stoichiometric mismatches (Denno & Fagan, 2003; Hes-
sen et al., 2013). These mismatches may hamper or prevent the
development of saproxylic species (Filipiak & Weiner, 2014;
Filipiak et al., 2016). Thus, it can be concluded that the content
of nutritional elements in dead wood is too low to produce
the biomass of a xylophage. Many wood-eating invertebrates
are nevertheless able to survive and, indeed, often thrive and
prosper on this low-quality food source. This can be achieved by
means of advanced and expensive adaptive traits (e.g. selective
feeding, overeating and selective absorption: Anderson et al.,
2005; Frost et al., 2005; Hessen & Anderson, 2008) for the
Correspondence: Michał Filipiak, Institute of Environmental Sci-
ences, ul. Gronostajowa 7, 30-387 Krakow, Poland. Tel.: +48 12 664
51 34; e-mail: michal0lipiak@gmail.com; michal.lipiak@uj.edu.pl
growth and maintenance of body tissues with proper chemical
composition. For dead wood, however, the mismatch appears to
be too severe to be overcome by these traits alone. The C:N and
C:P ratios in dead wood may reach approximately (element: dry
mass ratio/atomic ratio) N: 6500/7500 and P: 54 500/150 000
(Lambert et al., 1980; Harmon et al., 1986; Weedon et al., 2009;
Palviainen et al., 2010a, 2010b; Filipiak & Weiner, 2014; Köster
et al., 2015). This incompatibility must be mitigated irrespective
of the digestibility of the woody high-C components of the diet:
an extremely high C:other elements ratio of the food means that,
to extract the required amounts of elements other than carbon,
an organism must process an extraordinarily large mass of food
at the same time as excreting the superuous carbon-rich com-
pounds, without regard to their potential digestibility. Previous
studies concerning the adaptive traits required to overcome a
stoichiometric mismatch do not consider such a high excess of
C(Higashiet al., 1992; Darchambeau et al., 2003; Anderson
et al., 2005; Frost et al., 2005; Hessen & Anderson, 2008). The
action of microbial symbionts capable of digesting cellulose
may improve the digestibility of polysaccharides (Ljungdahl &
© 2016 The Royal Entomological Society 1
2M. Filipiak and J. Weiner
Eriksson, 1985; Douglas, 2009) and provide wood eaters with
additional nitrogen (Roskoski, 1980), although the required
net supply of nutritional elements other than C and N cannot
be increased in this way. Another solution may be ‘nutritional
supplementation’ of wood by ingrowing fungi that import the
matter needed to compose their tissues from the outside envi-
ronment. Calculations suggest that 85 years would be required
for a xylophage to accumulate the necessary amount of elements
to build its body from pure dead wood, whereas this process
takes only 3– 4 years in nature (Filipiak & Weiner, 2014). Accu-
mulating the elements from dead wood infected by nutritionally
rich fungal mycelia is consistent with this 3–4-year time frame
(Filipiak & Weiner, 2014) because the fungal import of non-C
elements results in a fundamental rearrangement of dead wood
stoichiometry during the initial phase of decay (Filipiak et al.,
2016). These considerations suggest that ‘dead-wood eaters’
rely neither on pure dead wood as their key food source, nor on
gut microbes as key nutrient deliverers. Nevertheless, to develop
to adulthood, the larvae of xylophagous beetles living in an
extremely nutritionally decient environment must accumulate
sufcient amounts of elements. To determine the mechanism by
which nutritional balance is maintained, two common species
of pine-xylem-feeding beetles are selected as model organisms:
Stictoleptura rubra L. and Chalcophora mariana Dejean. These
organisms occupy similar ecological niches (developing on
decaying pine wood and facing stoichiometric mismatch) but
belong to two different taxa (Cerambycidae and Buprestidae)
and differ in body size. The beetles inhabit pine stumps in
numbers that permit the collection of sufcient sample quan-
tities for chemical analysis. The maximum development time
ranges from 3 to 4 years for S. rubra to 6 years for C. mariana
(Dominik & Starzyk, 2004; Walczy´
nska, 2012). Over such a
long period of development, the process of wood decomposition
advances, and the concentration of nutrients increases. Over the
time corresponding to larval development of a xylophage, the
nutrient concentration may increase by 23-fold for N, 40-fold
for P, six-fold for Cu, six-fold for K and seven-fold for Fe as a
result of transport from the outside environment by ingrowing
fungi (Filipiak & Weiner, 2014; Filipiak et al., 2016). Thus,
xylophages develop in a nutritionally dynamic environment.
The present study aims: (i) to determine the changes in body
concentrations of nutritional elements (C, N, P, K, Na, Ca, Mg,
Fe, Zn, Mn and Cu) during the ontogenesis of xylophagous bee-
tles; (ii) to relate the ontogenetic dynamics of body composi-
tion to changes in the elemental composition of dead wood; and
(iii) to identify the strategy used by xylophages to overcome the
extreme nutritional poverty of dead wood.
Materials and methods
Two common species of pine xylem-feeding beetles were
used: S. rubra Linnaeus (=Corymbia rubra Nakano and
Obayashi, 1957 and Aredolpona rubra Viliers 1974) and
C. mariana Linnaeus (=Buprestis mariana Linnaeus 1758;
Coleoptera, Buprestidae). Both adult beetles and pine stumps
that most likely contained beetle larvae were collected in
the Niepołomice Forest (southern Poland, 50∘05′N, 20∘21′E,
elevation 184 –212 m.a.s.l.), in the spring, summer and autumn
of 2010–2012. Stumps aged 1 –4 years after tree logging were
hand split to collect larvae and pupae.
Raw data on the chemical composition of the wood, adult
beetles and pupae were obtained from a previous investigation
(Filipiak & Weiner, 2014) (see Supporting information, Tables
S1 and S2; larvae, pupae and adults were sampled at the same
sites and at the same time; the larvae investigated in the present
study inhabited the pine stumps described by Filipiak & Weiner,
2014). These data were used to compare the dynamic elemental
composition of growing beetles with the temporal stoichiometric
changes in their potential food resulting from ongoing decay
processes. The wood samples were classied by the degree of
decay in accordance with Esseen et al. (1997): (i) undecayed
wood – no visible changes caused by microorganisms, hard and
healthy; (ii) moderately decayed wood – considerably changed
by microorganisms, discolored (purple and dark brown), wet and
softer than (i) but still difcult to tear apart with a knife; and (iii)
highly decayed wood – many visible changes, ample layers of
white or brown rotting fungi, wet, soft and easily torn apart with
a knife or even by hand.
The larvae of both beetle species were divided into age classes
(L1–L3 for S. rubra and L1 –L4 for C. mariana) based on the
maximum breadth of the head capsule. Extreme values for head
capsule width were selected for the denition of classes based
on discontinuities in the width distribution (for both species).
A greater number of classes could not be distinguished because
small individuals were pooled to reach the minimum measurable
concentrations of elements in a sample. For S. rubra,theage
classes were determined from data obtained in previous exper-
iments (Walczy´
nska, 2010, 2012). The age classes used in the
present study were: L1 (head capsule width 0.35–2.80 mm),
up to 290 days; L2 (2.89 –3.66), from 290 to 620 days; and L3
(3.76–4.65), more than 620 days. There were no previous data
suitable for determining an approximate age for C. mariana lar-
vae. Therefore, age classes were dened arbitrarily according to
the head capsule widths: L1, 0.40 –2.17 mm; L2, 2.35 –2.68 mm;
L3, 2.90– 3.36 mm; and L4 3.48 – 9.24 mm. These four stages
cover the entire period of larval development (pupae and adults
were found in the most heavily decayed stumps, although larval
development may take as long as 6 years).
Insect and wood samples were freeze-dried to a constant
mass. Their C and N contents were then determined using an
automated analyzer (Vario EL III automatic CHNS analyzer;
Elementar Analysensysteme GmbH, Germany), whereas the
contents of K, Ca, Mg, Fe, Zn, Mn, Cu and Na were mea-
sured via atomic absorption spectrometry (Perkin Elmer AAn-
alyst 200 and AAS: PerkinElmer AAnalyst 800; Perkin Elmer
Inc., Waltham, Massachusetts) and the P content by colorime-
try (MLE Flow injection analyzer; Medizin- und Labortechnik
Engineering GmbH, Germany). Samples (one to several individ-
uals depending on size) were mineralized by acid digestion with
a solution of 4 : 1.5 :1 nitric acid (70%), perchloric acid (65%)
and sulphuric acid (95%) before analysis using atomic absorp-
tion spectrometry and ow injection colorimetry. Sulphanilic
acid was used as a reference material for C and N analyses. Cer-
tied reference materials were used for the other nine elements
(bush, NCS DC 733348; chicken, NCS ZC73016; and pork
© 2016 The Royal Entomological Society, Physiological Entomology, doi: 10.1111/phen.12168
Stoichiometry of xylophage development 3
muscle, NCS ZC 81001, NCS Testing Technology Co., Ltd.,
Beijing, China).
A principal component analysis (PCA) was used to compare
the multi-element stoichiometric relationships between develop-
mental stages. The data were log transformed, centred and stan-
dardized by species but not by sample (i.e. PCA was performed
on a correlation matrix; , version 4.5; Biometrics, Plant
Research International, The Netherlands). To test for signicant
differences between clusters in multidimensional space, which
represent larvae of the same developmental stage, independent
analyses of variance () was performed for the rst and
second axis scores (, version 10; StatSoft, Inc., Tulsa,
Oklahoma). A Kruskal–Wallis test was used to test for differ-
ences (P<0.05) between developmental stages in relation to the
nutritional composition (, version 10).
Trophic stoichiometric ratio (TSR)
The degree of stoichiometric mismatch between the larvae
and their food for each element, x, was expressed as the trophic
stoichiometric ratio (TSR), a modied version of the threshold
elemental ratio (TER) (Urabe & Watanabe, 1992; Sterner &
Elser, 2002; Fagan & Denno, 2004):
TERx=(GGEx∕GGEc)×(C∶X)i+1(1)
where GGExis gross growth efciency of the element x;GGEC
is gross growth efciency of carbon; iis trophic level; Cis
concentration of carbon; and Xis concentration of element x.
If:
(C∶X)i≥TERx(2)
then element xmay become a limiting factor for growth at
trophic level i+1. The gross growth efciencies for any given
element can be experimentally measured (e.g. through feeding
trials in growing animals, using foods with different contents of
the studied elements). However, such data are in fact extremely
scarce and are almost non-existent for elements other than N
and P. In practical terms, the TER index for invertebrates can
only be estimated using arbitrary assumptions (Fagan & Denno,
2004; Frost et al., 2006; Doi et al., 2010). Thus, there is a need
to develop a simpler index, based on readily available data,
to enable the detection of possible stoichiometric mismatches
between trophic levels and comparisons between various taxa,
habitats and foods. With the aim of estimating the degree of
possible stoichiometric mismatch based on existing data, a
simplied approach is proposed, rewriting condition (2):
(C∶X)i≥(GGEx∕GGEc)×(C∶X)i+1
to obtain:
(C∶X)i∕(C∶X)i+1≥GGEx∕GGEc
or TSRx≥GGEx∕GGEc
Even without precise information about elemental conversion
efciencies for a given organism, it can be safely assumed
that GGEx>GGEcbecause carbon, as a metabolic energy
substrate, is always partially lost as CO2. The actual values of
GGE for carbon and other elements depend on the growth rate,
the amount of each element available in food (C:Xratio), the
possible absorption efciency, and body mass (as a result of the
metabolic rate allometry). Without any information about these
relationships, it may only be assumed that the gross growth ef-
ciency for an essential nutrient may be maximized. At any rate,
the larger the difference between GGEXand GGEc, the more
severe is the trophic mismatch. Based on the limited amount
of published data (Fagan & Denno, 2004), it was assumed that
the minimum value of GGEcin invertebrates would not exceed
approximately 0.25, whereas the maximum possible GGExmay
approach 1 (because it is maximized, although its actual value
may be lower). Under these assumptions, the still balanced
threshold ratio (GGEx/GGEc) equals 1/0.25 =4.0. Assuming a
lower GGEc(e.g. 0.1, with 90% of assimilated carbon excreted
via respiration, which is unlikely), a threshold value of 10.0
would be obtained. With a lower actual GGExvalue (e.g. 0.5)
and GGEc=0.25, the threshold may be as low as 2.0; all of
these values are still within the same order of magnitude. To
obtain insight into the approximate stoichiometric mismatch
based on the available data, it can be arbitrarily and conserva-
tively assumed that for TSRx≥4.0, element xmay impose a
constraint on growth, with more severe mismatches indicated
by even higher values. TSR is not meant to represent the actual
measured TER of a given element but serves as a relative and
conservative index indicating a possible stoichiometric mis-
match. Various elements may be acquired, assimilated, reused
and excreted differently. The TSR index assumes that noncarbon
elements are assimilated from food into the body at a maximal
rate (100%). Thus, the actual mismatches in natural situations
may only be greater than the values estimated as TSR.
TSRxwas calculated as:
TSR =(C∶X)food ∕(C∶X)consumer
where Crepresents the carbon concentration, and Xrepresents
the concentration of element x.TheTSR index is not dependent
on the units used for the stoichiometric ratios of C:X(molar or
mass units).
The TSR index calculated in the present study is based on the
separately estimated chemical compositions of insects and their
potential food (decaying wood). The applied analytical proce-
dures require separate sample preparation for the determination
of C and N contents (dried tissue for CHNS analysis) and all
other elements studied (liquid product of acid digestion of tis-
sues for atomic absorption spectrometry and ow injection anal-
ysis). As a result of the small size of larvae and the difculty
of obtaining a perfect homogenate, the best way to obtain reli-
able results is to use whole specimens (or samples composed
of several specimens). As a consequence, the analytical results
for C and N contents were based on different specimens than
the results for other elements, and comparison of TSR indices
between various experimental groups was not possible based
on the variance of the individual TSRs. Instead, a randomiza-
tion procedure was applied and TSR values were calculated
from the C:Xratios of values randomly drawn from the dis-
tribution of the measured elemental contents for larval stages
© 2016 The Royal Entomological Society, Physiological Entomology, doi: 10.1111/phen.12168
4M. Filipiak and J. Weiner
L1 L2 L3 pu im
46
48
50
52
54
56
58
60
62
C %
ab
a
b
b
a
L1 L2 L3 pu im
4
6
8
10
12
N %
aa
a
a
b
L1 L2 L3 pu im
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
P %
a
abc
b
c
b
L1 L2 L3 pu im
2000
6000
10000
14000
18000
22000
K ppm
bb
a
ab
aa
aa
a
a
L1 L2 L3 pu im
0
200
400
600
800
1000
1200
1400
1600
Na ppm
a
b
a
ab ab
L1 L2 L3 pu im
500
1500
2500
3500
4500
5500
Mg ppm
a
a
b
ab
ab
L1 L2 L3 pu im
0
40
80
120
160
200
Fe ppm
ab
ab
b
a
b
L1 L2 L3 pu im
0
400
800
1200
1600
Ca ppm
L1 L2 L3 pu im
0
5
10
15
20
25
30
35
40
Mn ppm
aa
a
b
b
L1 L2 L3 pu im
20
60
100
140
180
220
260
Zn ppm
ab
a
b
aa
L1 L2 L3 pu im
0
10
20
30
40
50
60
70
80
90
Cu ppm
b
a
a
aab
(A) Stictoleptura rubra
Median
25%-75%
Min-Max
Fig. 1. Dynamics of elemental concentrations during ontogeny of two xylophagous beetle species. (A) Stictoleptura rubra.(B)Chalcophora mariana.
Stages of development. L1, L2, L3 (L4): larvae of different ages (from youngest to oldest); pu, pupae; im, imagines. The letters represent signicant
differences in the elemental composition between developmental stages (Kruskal– Wallis test, P<0.05) (n=3– 41, depending on stage of development
and element) (see Supporting information Table S1 for details and Table S2 for absolute elemental contents). The content of C increased quickly,
reached a maximum during the nal larval stages or pupal stage, and decreased in imagines, whereas the concentrations of other elements decreased
steadily and reached a minimum during the nal larval stages, then increased again in pupae and imagines.
L1–L4 and stages 1 –3 for decaying wood. The values were
simply drawn from the distribution of measured elemental con-
tents, without applying any bootstrapping technique (cf. Filipiak
& Weiner, 2014). The number of possible recombined TSR val-
ues in various groups may reach approximately 1000– 100 000,
from which 1000 TSR values for each C:Xratio were drawn.
From these distributions, the average values and 10th and 90th
percentiles were obtained.
Calculation of TSRs as a measure of the current stoichiometric
mismatch during larval development
The larval body composition differs between stages as a result
of the growth of new tissue (which may exhibit a different
chemical composition) and simultaneous losses (egestion,
moulting, and respiration). The stoichiometric balance should
also involve the net change in the absolute elemental content
in the increased body mass, compared with the amounts of
elements ingested during the same period of time.
Thus, the increase in dry mass was rst calculated between
two adjacent larval stages (Δm): Δm=m2–m1,wherem2is the
mean dry mass of the older larval stage and m1is the mean dry
mass of the younger larval stage. Then, the net increment of ele-
ment xduring the growth phase (Δx– absolute mass of element
xin Δm) was calculated as: Δx=m2×X2–m1×X1,whereX2
is the mean concentration of element xin the older larval stage,
and X1is the mean concentration of element xin the younger
larval stage. The ratio XΔ=Δx/Δmrepresents the apparent con-
centration of element xin the mass increment. Using this ratio,
© 2016 The Royal Entomological Society, Physiological Entomology, doi: 10.1111/phen.12168
Stoichiometry of xylophage development 5
(B) Chalcophora mariana
Median
25%-75%
Min-Max
L1 L2 L3 L4 pu im
46
48
50
52
54
56
58
60
C %
a
ab
ab
b
b
ab
L1 L2 L3 L4 pu im
3
4
5
6
7
8
9
10
11
N %
ab
a
ab
bb
b
L1 L2 L3 L4 pu im
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
P %
ab
a
b
aa
a
L1 L2 L3 L4 pu im
2000
6000
10000
14000
K ppm
aa
a
ab ab
b
L1 L2 L3 L4 pu im
0
400
800
1200
1600
2000
2400
Na ppm
ab
ab
a
ab
b
ab
L1 L2 L3 L4 pu im
600
1000
1400
1800
2200
2600
3000
Mg ppm
ab ab
b
a
bb
L1 L2 L3 L4 pu im
0
20
40
60
Fe ppm
ab
ab ab
a
a
b
L1 L2 L3 L4 pu im
0
5
10
15
20
25
30
35
40
45
Mn ppm
cc
cac
b
ab
L1 L2 L3 L4 pu im
10
30
50
70
90
110
Zn ppm
b
aa
a
a
ab
L1 L2 L3 L4 pu im
0
4
8
12
16
20
Cu ppm
aab ab b
a
a
L1 L2 L3 L4 pu im
0
200
400
600
800
1000
1200
Ca ppm
ab
ab ab
a
bb
Fig. 1. Continued.
the TSR values representing the stoichiometric balance during
the mass increment were calculated by applying the procedure
described earlier but using only the mean values of elemental
concentrations (without randomization). The dead wood cate-
gories described earlier and the average values for the two adja-
cent dead wood categories (undecayed/moderately decayed and
moderately decayed/highly decayed) were employed as food
sources. The means were used as an approximation of the chang-
ing stoichiometry of the food during larval development.
Results
Changes in the elemental composition of larvae during
development
The percentage of C in the bodies of both beetle species
increased signicantly during larval development, peaking in
the oldest larvae and pupae, followed by a decrease after eclo-
sion (Fig. 1). Other elements showed a reverse pattern (Fig. 1).
Certain elements (P and K in both species and Na and Mg in
S. rubra) exhibited the highest average concentrations during
the rst stage of larval development, although the variations
in elemental contents within each developmental stage were
high, and the differences between stages were not signicant
in most cases (Fig. 1). The total data on relative and absolute
elemental contents are presented in Supporting information,
Tables S1 and S2. In both species, the patterns of changes in the
concentrations of elements during development were similar.
Dynamics of xylophage multi-elemental stoichiometry
Principal component analysis of the concentrations of all stud-
ied elements in the larvae, pupae and imagines revealed similar
patterns of stoichiometric changes in the body composition
© 2016 The Royal Entomological Society, Physiological Entomology, doi: 10.1111/phen.12168
6M. Filipiak and J. Weiner
Tabl e 1 . Values of the rst two principal component loadings for the principal component analysis plots in Fig. 2 for the two xylophagous species: Stictoleptura
rubra and Chalcophora mariana.
Axis C N P K Na Mg Fe Ca Mn Zn Cu
Stictoleptura rubra
Axis 1 0.64 −0.95 −0.31 0.43 −0.66 0.47 −0.93 −0.35 −0.78 −0.81 −0.82
Axis 2 −0.52 −0.01 0.86 0.87 0.38 0.82 0.12 0.29 −0.56 0.40 −0.23
Chalcophora mariana
Axis 1 0.58 −0.89 −0.80 −0.67 −0.68 −0.85 −0.90 −0.93 −0.69 −0.95 −0.88
Axis 2 −0.62 −0.21 0.48 0.61 −0.12 0.06 −0.20 −0.25 −0.63 0.04 −0.15
Fig. 2. Multivariate analysis of changes in body composition during ontogenesis in the two studied beetle species, Stictoleptura rubra (A) and
Chalcophora mariana (B), based on 11 elements: principal component analysis (PCA) plots (rst two axes). L1– L4: larval stages (L1 is the youngest,
L3 or L4 is the oldest). An individual dot represents one sample, n=71 for S. rubra and 79 for C. mariana. Larval stages differ mainly as a result of
increases in the C concentration and decreases in P, K and Mg concentrations with age. Non-C elements are concentrated in adults (for the values of
the loadings, see Table 1; for statistical analysis of score values, see Fig. 3).
during ontogenesis for the two species. The rst two principal
components explained 80.4% and 76.0% of the observed vari-
ance in elemental concentrations for C. mariana and S. rubra,
respectively, with a prevailing contribution of the rst axis,
conrming a strong correlation between these variables.The
rst principal component primarily explained the variance in
N, Fe, Cu and Zn contents, and the second principal compo-
nent explained primarily the variance in K, P and C contents,
although there were differences between species, especially
for P and Mg (Table 1). The PCA plots indicated that each
developmental stage exhibited a specic multi-elemental stoi-
chiometry and that, in both species, it proceeded from one stage
to another along a similar trajectory in multidimensional space
(Figs 2 and 3). The transition from the rst to the nal stage of
larval development in S. rubra proceeded along the second axis,
loaded mostly by P, K, Mg and C (Fig. 2 and Table 1), whereas,
in C. mariana, the same trajectory ran diagonally, determined
mostly by P, K and C (Fig. 2 and Table 1). The transition from
the nal larval stage to pupae and adults in both species pro-
ceeded along the rst axis (Fig. 2), with a similar contribution
of all elements (Table 1).The clusters of individuals belonging
to the same larval classes differed from each other in both
species, and all larval classes differed from the pupae and adults
in both species, whereas the adult and pupal clusters overlapped
(Fig. 2).
The s calculated for the PCA scores (Fig. 3) indicate
signicant stoichiometric differences between all developmen-
tal stages for S. rubra and between the three (a, b and
c) stoichiometrically similar groups in C. mariana: (a) the
rst three larval stages (L1, L2, L3); (b) the oldest larval
stage (L4); and (c) the pupae and imagines. In both species,
the adults (after eclosion) exhibited higher concentrations of
all elements except C, although this pattern was less obvi-
ous for K and Mg in S. rubra. These nutrients appeared to
be highly concentrated in the youngest larvae (L1 and L2)
(Fig. 2).
Changes in the elemental composition of wood during larval
development
The elemental composition of wood inhabited by larvae
changed during larval development. The initial contents of
elements other than C were low and increased considerably
during the rst 4 years after tree cutting (Fig. 4), a period
corresponding to the developmental time of the larvae of the
studied species. The relative increments of the concentrations
during wood decay were highest for N (23-fold), P (14-fold),
Cu (6.3-fold) and K (four-fold), as also previously shown by
Filipiak & Weiner (2014). According to the PCA results, the
© 2016 The Royal Entomological Society, Physiological Entomology, doi: 10.1111/phen.12168
Stoichiometry of xylophage development 7
L1 L2 L3 pupae imagines
–1.5
–1.0
–0.5
0.0
0.5
1.0
1.5
2.0
1st PC - scores
Stictoleptura rubra
a
b
c
d
e
L1 L2 L3 pupae imagines
–1.5
–1.0
–0.5
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
2nd PC - scores
a
b
cc
c
(C)
(D)
L1 L2 L3 L4 pupae imagines
–2.0
–1.5
–1.0
–0.5
0.0
0.5
1.0
1.5
1st PC - scores
ab
aa
c
ab b
Chalcophora mariana
L1 L2 L3 L4 pupae imagines
–2.0
–1.5
–1.0
–0.5
0.0
0.5
1.0
1.5
2.0
2.5
2nd PC - scores
aaa
c
bb
(A)
(B)
Fig. 3. The rst two principal component scores for the elemental concentrations plotted for the developmental stages of Chalcophora mariana and
Stictoleptura rubra. Error bars denote the SE. Values with different letters indicate signicant differences in the elemental composition between larval
stages, pupae and imagines [analysis of variance (), unequal n, honestly signicant difference test, P<0.05]. (A) Chalcophora mariana,
for the scores of the rst principal component (F6,76 =91.919, P=0.0000). (B) Chalcophora mariana, for the scores of the second principal
component (F5,86 =97.766, P=0.0000). (C) Stictoleptura rubra, for the scores of the rst principal component (F4,77 =242.25, P=0.0000).
(D) Stictoleptura rubra; for scores of the second principal component (F4,77 =43.711, P=0.0000).
-1.0 1.0
-1.0
1.0
C
N
P
K
Na
Ca
Mg
Fe
Zn
Mn
Cu
Axis 1 (47.49%)
Axis 2 (12.5%)
Undecayed Moderately
decayed
Highly
decayed
Fig. 4. Changes in the stoichiometry of dead wood inhabited by
xylophagous beetles: principal component analysis (PCA) plot (rst two
axes). The wood samples were classied by the degree of decay. Data are
from Filipiak & Weiner (2014). An individual dot represents one sample
(n=37). Elements that were most limiting for xylophage development
showed the greatest increase in concentrations with decay (relative to
other elements; for values of loadings, see Table 2).
Tabl e 2 . Values of the rst two principal component loadings for the
principal component analysis plots in Fig. 4, with K, P and N showing the
highest relative supplementation rate in decaying dead wood.
Axis C N P K Na Mg Fe Ca Mn Zn Cu
Axis 1 −0.75 0.81 0.85 0.86 0.57 0.60 0.58 0.74 0.52 0.56 0.63
Axis 2 0.47 0.08 −0.03 0.09 0.11 0.55 −0.40 0.26 0.54 −0.12 −0.54
rst two axes represented 60% of the observed variation, with
a prevailing contribution of the rst axis (Table 2), indicating a
strong correlation of all elemental contents (Fig. 4) and showing
that the clusters representing samples from the nal stage of
wood decay were shifted from the initial stage along the rst
axis. The loading of the rst axis was dominated by K, P,
N, Ca and C (loadings >0.74) (Table 2). The relatively high
contribution of C, Mg, Mn and Cu to the loading of the second
axis (Table 2) caused translocation of the cluster represent-
ing moderately decayed wood samples along the second axis
(Fig. 4).
Changes in nutritional mismatches during larval development
The nutritional mismatch between the studied larvae and
their food varied according to larval age and the corresponding
decay stage of the dead wood (Fig. 5; for detailed data on all
combinations of development and wood decay stages, see also
Supporting information, Table S3). The mismatches, expressed
as TSR values (Fig. 5), were more pronounced for undecayed
wood and the youngest larvae and decreased with the wood
© 2016 The Royal Entomological Society, Physiological Entomology, doi: 10.1111/phen.12168
8M. Filipiak and J. Weiner
N
P
K
Na
Mg
Fe
Ca
Zn
Mn
Cu
0.01
0.1
1
10
100
1000
10000
4
wood decay categories
larvae age clasess
L1/Undecayed L2/Moderately L3/Highly
less decayed
(A) (B)
more decayed
younger older
wood decay categories
larvae age clasess
less decayed more decayed
younger older
TSR ratio (C:x food/C:x larvae)
Stictoleptura rubra Chalcophora mariana
L1/Undecayed L2/Moderately L4/Highly
Fig. 5. Changes in trophic stoichiometric ratio (TSR) values during larval development of Stictoleptura rubra (A) and Chalcophora mariana (B)
(TSRx=(C:X)wood/(C:X)larvae , where C represents the content of carbon, and Xrepresents the content of element xfor larvae and their potential food).
The TSR ratios were calculated for (i) L1 larvae and undecayed wood; (ii) L2 larvae and moderately decayed wood; and (iii) L3 (S. rubra)orL4(C.
mariana) larvae and highly decayed wood. TSR values above 4 denote limitations on development. The dotted line indicates the threshold value of
TSR =4 (detailed results, including calculations for all of the larval stage/wood decay categories, are provided in the Supporting information, Table
S3). In general, the development of xylophages is co-limited by the scarcity of N, P, K, Na, Mg, Cu and Zn, although the limitation decreases along the
dead wood decay gradient corresponding to the larval growth of the xylophages.
Tabl e 3 . Estimated elemental composition of growing tissue during larval development of Stictoleptura rubra and Chalcophora mariana.
C N P K Na Ca Mg Fe Zn Mn Cu
Adjacent larval stages (%) (ppm)
Stictoleptura rubra
L1 First stage 50.1 7.22 0.92 17 539 762 825 3977 61.6 140 3.00 8.62
X𝚫L2– L1 54.0 6.66 0.56 11 136 377 405 2704 38.0 58.1 3.67 13.6
L3– L2 54.4 6.61 0.53 10 540 342 366 2586 35.8 50.4 3.73 14.1
L3 Last stage 58.2 5.37 0.51 8640 382 339 1915 22.9 60.3 7.86 9.77
Chalcophora mariana
L1 First stage 49.1 6.70 0.73 10 473 585 438 1979 34.5 81.4 4.60 5.81
X𝚫L2– L1 52.0 5.55 0.71 9800 489 246 1907 13.6 54.8 4.47 4.23
L3– L2 55.8 3.96 0.67 10 970 834 350 2412 16.1 76.5 8.68 4.91
L4– L3 58.2 3.70 0.32 5036 342 75.8 1082 9.8 29.7 6.59 3.05
L4 Last stage 56.7 4.09 0.44 6647 433 147 1398 11.9 40.9 6.49 3.55
XΔis the concentration of element xin the body mass increment between two adjacent larval stages. Adjacent larval stages are larval age classes between which
the tissue was growing (an explanation of the calculation is provided in the text); rst stage is the youngest age class measured directly (L1, concentrations
of elements given for the mean whole body); last stage is the oldest age class measured directly (L3 or L4, concentrations of elements referred to the mean
whole-body values).
decay gradient and larval age. The TSR values indicated that
the development of S. rubra was co-limited by the scarcity of
N, P, K, Na, Mg, Cu, Zn and Fe and that the development of
C. mariana was co-limited by the scarcity of N, P, K, Na, Mg,
Cu and Zn (Fig. 5; see also Supporting information, Table S3).
The most striking mismatches (1–3 orders of magnitude) were
observed for N, P, K, Na and Mg. Cu tended to be limiting in
all larval classes of S. rubra and all wood categories, although
only in L1 and undecayed wood in the case of C. mariana.Zn
was limiting in undecayed wood and moderately decayed wood
and in the L1 and L2 larvae of both species. Fe was limiting in
S. rubra L1 larvae and undecayed wood. Only Ca and Mn were
not limiting nutritional elements (they are available in excess in
the food, Fig. 5; see also Supporting information, Table S3).
The relationship between the mismatches for N and P changed
with the wood decay gradient from more severe limitation by N
in the case of undecayed dead wood and the youngest larvae to
more severe limitation by P or similar limitation by N and P in
the case of highly decayed wood and the oldest larvae (Fig. 5;
see also Supporting information, Table S3). A change in the
relationship between the TSR values was also observable for K
and Na, as well as for Zn and Cu. The value for K was higher than
that for Na in undecayed wood and the youngest larvae, whereas
the value for K was lower than that for Na in the oldest larvae
and highly decayed wood. The value for Cu was higher than that
for Zn in the youngest larvae and undecayed wood, whereas the
value for Cu was similar to or lower than that for Zn in the oldest
larvae and highly decayed wood (Fig. 5).
© 2016 The Royal Entomological Society, Physiological Entomology, doi: 10.1111/phen.12168
Stoichiometry of xylophage development 9
Tabl e 4 . Trophic stoichiometric ratio (TSR) values reecting stoichiometric
mismatches experienced by xylophage larvae of Stictoleptura rubra and
Chalcophora mariana during development.
Larval
stage
Wood
category NPKNaCaMgFeZnMnCu
Stictoleptura rubra
L1 Undec. 935 1006 130 51 0.92 37.5 4.9 16 0.06 24
L2– L1 Mod.-undec. 287 172 52 23 0.39 22.2 1.7 5.5 0.07 7.9
L3– L2 High.-mod. 48 48 19 17 0.29 14.9 1.1 4.1 0.06 4.4
L3 High. 21 28 10 14 0.22 8.2 0.59 4.3 0.10 2.8
Chalcophora mariana
L1 Undec. 885 810 79 40 0.50 19.0 2.8 9.2 0.09 16
L2– L1 Mod.-undec. 248 226 47 31 0.25 16.3 0.64 5.4 0.09 2.5
L3– L2 Mod.-undec. 165 197 49 50 0.33 19.2 0.71 7.0 0.15 2.7
L3– L2 High.-mod. 28 58 19 39 0.27 13.5 0.47 6.0 0.13 1.5
L4– L3 High.-mod. 25 27 8.5 16 0.06 5.8 0.27 2.2 0.10 0.89
L4 High. 16 25 7.8 17 0.10 6.1 0.31 3.0 0.08 1.1
Wood categories: undec., undecayed; mod., moderately decayed; high., highly decayed.
For in-between categories of wood and larval stages, the mean was calculated from
the two adjacent categories (for explanation, see text). Values >4 are shown in bold,
indicating the nutritional limitation of the development of xylophages.
Current stoichiometric mismatches experienced during
development
InthecaseofS. rubra, the stoichiometry of body mass (Δm)
that was built between two adjacent larval stages was stable
throughout development and differed from the stoichiometry of
the whole larvae (Table 3). In particular, the Cu concentration in
Δmwas relatively high, whereas the Zn and Fe concentrations
relatively low compared with the concentrations measured in the
bodies of the youngest larvae (L1). The Δmwas also enriched
in C (most likely because of fat deposition). In C. mariana,
Δmvaried with age (Table 3). In this species, the C content
increased with increasing larval age, whereas the concentrations
of other nutritional elements were lower, showing a gradient
of changes from low– C, high–other elemental concentrations
in L1 larvae to high– C, low–other elemental concentrations in
L4 larvae. The TSRs calculated for Δm(Table 4) showed that
the current stoichiometric mismatches experienced by growing
xylophages (Table 3) tended to be slightly less pronounced than
the mismatches calculated for the whole larval bodies (Fig. 5).
However, they were still extremely high and depended more
on the dead wood decomposition stage than on the larval age.
Thus, all of the results reported earlier based on the whole-body
stoichiometry remain valid. The TSR values calculated for XΔ
(concentrations of elements in the body mass built between two
adjacent larval stages) indicate that the development of S. rubra
was co-limited by the scarcity of N, P, K, Na, Mg, Cu, Zn and Fe
and that the development of C. mariana was co-limited by the
scarcity of N, P, K, Na, Mg, Cu and Zn (Table 4).
Discussion
Changes in body stoichiometry during xylophage development
In both species, the C concentration increases with age and
decreases after eclosion. This pattern is most likely attributable
to intense fat deposition during larval growth and the subsequent
exploitation of these reserves during the energetically costly
morphological transformations that occur during the pupal
stage. Growing tissue is also enriched in Cu (Table 3), partic-
ularly in S. rubra, which is more limited by the scarcity of this
element than C. mariana. In both species, the concentrations of
K,FeandZningrowingtissuetendtodecreasewithlarvalage
(Table 3). Almost all elements other than C (with the exception
of K and Mg in S. rubra and K in C. mariana) (Figs 1 and 2)
exhibit increased concentrations in adults during pupation. The
contents of three elements are exceptionally high in adults: Mn
in both species, Cu in S. rubra and Ca in C. mariana. A trend
of higher P concentration in younger larvae is observed in both
species (particularly in S. rubra) (Figs 1 and 2). According
to the growth rate hypothesis, an increased P concentration is
related to faster growth, and the atomic N : P ratio can be used
as a measure of the P demand for growth (Elser et al., 1996,
2000, 2003). The mean N : P atomic ratios for all larval stages
of both species initially increase in S. rubra (L1 N : P =6.7, L2
N:P=9.1, L3 N : P =9:1; L1 =the youngest, L3 =the oldest
larvae) and remain almost constant in C. mariana (L1 N : P =7.9,
L2 N : P =6.9, L3 N : P =7.2, L4 N : P =8.0; L1 =the youngest,
L4 =the oldest larvae). Back & King (2013) note that the N : P
ratio generally increases during development in aquatic insects
in association with a decreasing growth rate. The two species of
xylophage that are investigated in the present study may exhibit
different life histories: S. rubra may display rapid growth of the
youngest larvae, whereas C. mariana may maintain a relatively
constant growth rate throughout larval development.
Nutritional mismatches during larval development
During its lifetime, a xylophagous insect larva must be able
to obtain all of the elements required for growth. Dead wood
is extremely decient in all of these important elements except
C; however, the wood is not nutritionally stable, and a decrease
in C : X ratios is observed during the rst 4 years of decom-
position (Fig. 4). This nutritional rearrangement of dead wood
is a result of the development of a network of fungal connec-
tions between the soil and dead wood and the fungal ability to
mobilize nutrients from the soil into stumps (Swift et al., 1979;
Dighton, 2003; Filipiak & Weiner, 2014; Filipiak et al., 2016).
Fungi may connect dead wood stumps with patches of forest
oor that are nutritionally rich (decomposing matter, mineral
soil, bedrock rich in specic elements) and translocate nutri-
tional elements from these patches to the stumps (Stark, 1972;
Swift et al., 1979; Lodge, 1987; Boddy & Watkinson, 1995;
Dighton, 2003, 2007; Cairney, 2005; Watkinson et al., 2006;
Clinton et al., 2009; Mooshammer et al., 2014; Filipiak et al.,
2016). This nutritional rearrangement is the main factor respon-
sible for the observed mitigation of stoichiometric mismatches
experienced by growing larvae. The mismatches depend pri-
marily on the stage of dead wood decay (not on the larval ele-
mental composition; Fig. 5 and Table 4; for details, see also
Supporting information, Table S3). Undecayed dead wood (least
infected by fungi; Filipiak et al., 2016) presents xylophages with
stoichiometric mismatches that are too high to be overcome
© 2016 The Royal Entomological Society, Physiological Entomology, doi: 10.1111/phen.12168
10 M. Filipiak and J. Weiner
(Fig. 5 and Table 4). Larval development is limited by N and
P deciencies in food, as suggested by Lemoine et al. (2014),
as well as the scarcity of some minor nutrients, among which
K, Na, Mg, Zn, Fe and Cu represent limiting factors (Fig. 5
and Table 3; see also Supporting information, Table S3). The
limitation is mitigated during the decomposition of dead wood,
although large stoichiometric mismatches remain for ve ele-
ments (N, P, K, Na, Mg) (Fig. 5). These mismatches constrain
larval growth, resulting in prolongation of the larval develop-
ment time. The question arises: is it possible for xylophages to
overcome extreme stoichiometric mismatches in any other way
than relying on fungal import? Hessen & Anderson (2008) note
that an excess of C may be utilized to enhance tness. They also
describe mechanisms used to adjust to leftover C. However, they
only consider relatively low levels of C excess (the highest C : P
atomic ratio noted is 600, in contrast to the maximum value of
150 000 that may occur in dead wood). The extreme mismatches
observed in the present study cannot be mitigated by employing
simple strategies such as releasing gaseous CO2or increasing
body temperature and metabolic rate. In the case of an extreme
excesses of C in relation to other nutritional elements, the pre-
vailing mass of C must be excreted by defecation, regardless of
its potential digestibility (thus, the larvae must overfeed, which
in turn may cause a decrease in nutrient absorption). Indeed,
the C concentration in the frass of saproxylic insects is simi-
lar to that in wood (Chen & Forschler, 2016). Digestive tract
symbionts can improve the digestibility of food, supplement the
diet with N and synthesize important organic compounds from
nutrients furnished by the preliminary food or eliminate tox-
ins (Douglas, 2009, 2011; Feldhaar, 2011; Karasov et al., 2011;
Hammer & Bowers, 2015). However, considering the law of
conservation of mass, the content of any element processed by
microbial symbionts cannot be changed during any chemical
reaction performed by the microbes. Thus, although digestive
tract symbionts can decrease N limitation in xylophages and
do convert organic compounds, it is impossible for these sym-
bionts to have a role in decreasing limitations resulting from the
scarcity of multiple elements in food. Detritivores may over-
come dietary deciencies by feeding on microbes that cover
the detritus and concentrate nutrients (Horvathova et al., 2016).
Similarly, xylophages may cope with extreme stoichiometric
mismatches by relying on nutritionally rich fungal tissue as a
food source instead of dead wood (Filipiak & Weiner, 2014; Fil-
ipiak et al., 2016). Thus, the strategy used by xylophages to over-
come the extreme nutritional poverty of dead wood comprises
(i) relying on fungal rearrangement of dead wood stoichiometry
during the rst years of decay, disregarding changes in the body
stoichiometry of growing larvae, and (ii) prolongation of growth
supported by low mortality risk (Walczy ´
nska, 2010, 2012) and
good conditions for overwintering created by dead wood (Wal-
czy´
nska & Kapusta, 2016).
Roles of fungi in balancing the diet of xylophages inhabiting
dead wood and in food-web dynamics
Understanding the interactions of wood-eating beetles and
fungi appears to be important for understanding the wood
decomposition process and nutrient cycling in forests (Floren
et al., 2015). Klironomos & Hart (2001) describe how fungi
inuence food-web dynamics by supplying trees with N translo-
cated from previously predated animals in exchange for the
plant’s carbon. Fungi redistribute C between trees (Klein et al.,
2016). It is clear that not only C and N, but also other limit-
ing elements may be withdrawn by fungi from any source that
is impossible for other organisms to reach. The life histories
of xylophages are co-limited by the complex of nutritional ele-
ments required for maintaining their bodies’ multi-elemental
stoichiometry and for performing all physiological processes.
Fungi enrich dead wood in those nutritional elements that are the
most limiting for xylophage development, specically P, N, K,
Cu, Na, Mg, Zn and Fe (Filipiak & Weiner, 2014; Filipiak et al.,
2016). The present study claries the dependence of xylophages
on the action of fungi: xylophagous life cycles are shaped by the
import of N, P, K, Na, Mg and Cu into the nutritionally extremely
harsh environment of dead wood by fungi at the beginning of the
decay process. As the process proceeds, dead wood becomes a
more nutritionally balanced nourishment for xylophages. These
changes enable xylophages to grow an adequate mass of tis-
sue with an appropriate stoichiometry. Wood-feeding insects are
ecosystem engineers that change the nutritional properties of
dead wood, making the nutritive elements in dead wood avail-
able to other organisms via the insects’ frass (Chen & Forschler,
2016). Thus, fungi allow xylophages to develop, and xylophages
in turn affect dead wood, contributing to wood decomposition
and nutrient cycling in the forest oor.
Supporting Information
Additional Supporting Information may be found in the
online version of this article under the DOI reference:
DOI: 10.1111/phen.12168
Tabl e S1. Relative elemental contents in the studied beetle
larvae.
Tabl e S2. Absolute elemental contents in the studied beetle
larvae.
Tabl e S3. Trophic stoichiometric ratios (TSRX=
(C:X)wood /(C:X)larvae).
Acknowledgements
We are indebted to Ulf Bauchinger and the anonymous reviewers
for their constructive critical comments. The present study was
supported by the Polish Ministry of Science and Higher Edu-
cation (Grant No. DS/WBiNoZ/INo´
S/DS 756) and the National
Science Centre (Grant No. UMO-2011/01/B/NZ8/00103). We
thank Nadle´
snictwo Niepołomice (Niepołomice Forest Inspec-
torate) for support during the eld portion of the study. We also
thank Maciej Choczy´
nski, Paweł Dudzik and Patrycja Gibas for
assistance with the chemical analyses. Additionally, we thank
American Journal Experts (AJE) for English-language editing
of the manuscript submitted for publication.
© 2016 The Royal Entomological Society, Physiological Entomology, doi: 10.1111/phen.12168
Stoichiometry of xylophage development 11
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Accepted 21 August 2016
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