Journal of Animal Ecology 2010, 79, 923–931
doi: 10.1111/j.1365-2656.2010.01681.x
Root herbivores and detritivores shape above-ground
multitrophic assemblage through plant-mediated effects
Adela González Megı́as1* and Caroline Müller2
1
Department Biologı´a Animal, Facultad de Ciencias, Universidad de Granada, Spain; and 2Department of Chemical
Ecology, Faculty of Biology, Bielefeld University, Germany
Summary
1. Indirect effects mediated by changes in plant traits are the main mechanism by which aboveand below-ground herbivores affect each other and their enemies. Only recently the role of decomposers in the regulation of such plant-based systems has been considered. We hypothesized that: (i)
below-ground organisms, both herbivores (negative effect on plants) and detritivores (positive
effect on plants), will have a profound effect on the interactions among above-ground arthropods;
(ii) floral herbivores will negatively affect other above-ground herbivores associated with the plant;
and (iii) not only above- and below-ground herbivores, but also detritivores will affect the production of secondary metabolites, i.e. glucosinolates, in the plants.
2. We manipulated the presence of above-ground herbivores, below-ground herbivores and
below-ground detritivores on the Brassicaceae Moricandia moricandioides in the field to disentangle their individual and combined effects on other organism groups. We also investigated their
effects on the plant’s chemical defence to evaluate potential mechanisms.
3. Our results show that not only above- and below-ground herbivores, but also detritivores
affected other herbivores and parasitoids associated with the host plant. Most effects were not
additive because their strength changed when other organisms belonging to different functional
groups or food web compartments were present. Moreover, below-ground herbivore and detritivore effects on above-ground fauna were related to changes in glucosinolate concentrations and in
quantity of resources.
4. This study indicates that multitrophic interactions in plant-based food webs can dramatically
change by the action of below-ground organisms. One of the most important and novel results is
that detritivores induced changes in plant metabolites, modifying the quality and attractiveness of
plants to herbivores and parasitoids under field conditions.
Key-words: above–belowground interactions, multitrophic interactions,
responses, trait-mediated and density-mediated indirect interactions
Introduction
Ecological terrestrial communities are characterized by their
complex structures, which result from the variation in organism sizes, trophic groups and type of interactions in which
organisms are involved (Tscharntke & Hawkins 2002).
Recently, the importance of indirect interactions, and more
specifically of trait-mediated indirect interactions (TMII), as
determinants of the structure of ecological communities has
been highlighted (Abrams et al. 1996; Ogushi 2005; Ogushi,
Craig & Price 2007). Trait-mediated indirect interactions
involve effects transmitted from one species to another
through one or more intermediate species, involving changes
*Correspondence author. E-mail: [email protected]
plant-induced
in the phenotype of the interacting species (Werner & Peacor
2003; Schmitz, Krivan & Ovadia 2004; Ogushi 2005).
In terrestrial ecosystems, plants act as a conduit between
above- and below-ground levels, allowing above-ground herbivores to affect below-ground herbivores indirectly and vice
versa (Scheu 2001; Bardgett & Wardle 2003; Porazinska et al.
2003; Schröter et al. 2004; Wardle et al.2004; Kaplan et al.
2008). Therefore, the importance of indirect plant-mediated
interactions between these organism groups has been highlighted (van Dam et al. 2003; Bezemer & van Dam 2005;
Ogushi et al. 2007). The mechanisms behind plant-mediated
interactions between herbivores are diverse, whereas the
induction of plant chemical defences plays a crucial role
(Agrawal 1998; Bezemer & van Dam 2005; Denno & Kaplan
2007; Ogushi et al. 2007; Kaplan et al. 2008; Hopkins, van
Dam & van Loon 2009). Changes in the quality and quantity
2010 The Authors. Journal compilation 2010 British Ecological Society
924 A. González-Megı´as & C. Müller
of metabolites can affect both generalist and specialist herbivores by influencing not only their host-plant acceptance and
detoxification ability but also the foraging behaviour of their
predators and parasitoids (Scheu 2001; Bezemer et al. 2005;
Soler et al. 2005; Wolfe, Husband & Klironomos 2005; Rasmann & Turlings 2007; Hopkins et al. 2009).
Plant-centred indirect interaction webs not only involve
herbivores and their predators, but also other organisms such
as ants or mycorrhizal species can have an important role in
shaping the structure of ecological communities (Ogushi
et al. 2007). Furthermore, decomposers are prominent members in most food webs, interacting with plants and potentially affecting other organisms of the community (Scheu
2001; Bardgett & Wardle 2003; Porazinska et al. 2003; Schröter et al. 2004; Poveda et al. 2005). Only recently the role of
decomposers in the regulation of these types of systems has
been considered (Bonkowski et al. 2001; Bezemer & van
Dam 2005; Blouin et al. 2005; Poveda et al. 2005; Wolfe
et al. 2005). It is well-known that their activity enhances
microbial turnover, nutrient recycling and the breakdown of
organic matter, favouring plants in many ways (Bardgett &
Wardle 2003; Bardgett, Usher & Hopkins 2005). However,
only a few studies have explored their effects on other aboveor below-ground members of the community, which are mediated by their influences on plant performance, phenotype
and chemical defence (Wurst, Dugassa-Gobena & Scheu
2004a; Wurst et al. 2004b, 2006; Blouin et al. 2005; Poveda
et al. 2005; Lohmann, Scheu & Müller 2009).
Brassicaceae are well-characterized for their specific
defence system, the glucosinolate (GS)–myrosinase system
(Halkier & Gershenzon 2006). Root- as well as shoot-feeding
herbivores can alter the GS concentrations in both aboveand below-ground tissue, locally and systemically, and
thereby influence other members of the multitrophic system,
including predators and parasitoids (Hopkins et al. 2009).
Even detritivores can influence the GS and myrosinase concentrations of above-ground tissue (Lohmann et al. 2009).
Therefore, species of this plant family offer useful models to
study plant-mediated interactions between organisms, which
may be related to induce changes in plant chemistry.
In this study, we examine the role of above- and belowground multitrophic interactions in the regulation of a plantbased food web. We hypothesized that: (i) below-ground
organisms, both herbivores (negative effect on plants) and
detritivores (positive effect on plants), will have a profound
effect on the interactions among above-ground arthropods;
(ii) floral herbivores (FH) will negatively affect other aboveground herbivores associated with the plant; and (iii) not
only above- and below-ground herbivores, but also detritivores will affect the production of GS in the plants. To test
our hypotheses, we manipulated the presence of aboveground herbivores, below-ground herbivores and belowground detritivores in the field. We tested their isolated and
combined effects on other co-occurring mutualistic and
antagonistic organisms using Moricandia moricandioides
(Boiss.) Heywood (Brassicaceae) as a host. We also evaluated
the isolated and combined effects of the three manipulated
organism groups on the plant’s chemical defence, trying to
discern potential mechanisms underlying animal–plant–animal interactions under natural conditions, and to understand
the importance of TMII in shaping the food web structure.
Materials and methods
STUDY SYSTEM
The study was conducted in 2007 at Barranco del Espartal, a seasonal
watercourse located in the arid Guadix-Baza Basin (Granada, southeastern Spain).The climate is continental Mediterranean with strong
temperature fluctuations (ranging from )14 to 40 C) and high seasonality (hot summers and cold winters). The vegetation is an arid
open shrub-steppe dominated by Artemisia herba-alba Asso, A. barrelieri Bess., Salsola oppositifolia Desf., Stipa tenacissima L., Lygeum
spartum L. and Retama sphaerocarpa L. Furthermore, the annual
Brassicaceae species M. moricandioides is highly abundant in this
habitat and was used as a model system.
The insect community associated with this plant includes FH,
mainly Pontia daplidice L. and Euchloe crameri Batler (Pieridae) and
the diamondback moth (Plutella xylostella L., Plutellidae). Many
lepidopteran species also feed on the leaves, such as Pieris rapae L.,
Pieris brassicae L. (Pieridae) and P. xylostella (Gómez 1996).
EXPERIMENTAL SET-UP
During the winter of 2006–2007, undamaged seedlings of M. moricandioides were grubbed out from the same population the field, taking care of preserving the root systems. These seedlings were potted
in plastic pots (7 · 10 cm). Seedlings with no more than three young
developed leaves were selected to ensure similar age of all plants.
Plants were kept in a common garden until the beginning of spring
(end of March 2007) when they were moved back to the field. Plants
did not differ at that time point in number of leaves (8Æ24 ± 0Æ27
leaves, mean ± SE) or height (3Æ94 ± 0Æ40 cm) and none of the
plants had developed a flowering stalk. Once in the field, 120 plants
were distributed at random into three blocks approximately 100 m
apart (40 plants were assigned to each block). Plants were located in
five rows of eight plants, each plant 50 cm apart. Plants were re-potted using mixed soil (free of macroarthropods) from the study sites.
The new pots consisted of fibre–glass mesh cylinders (10 · 15 cm) of
1-mm mesh size to inhibit the entrance or escape of macroinvertebrates. These pots were then buried with the upper surface even with
the ground.
A full factorial design was used to test the effects of three factors
with two levels per factor, presence (+) or absence ()), on plant–insect
interactions. The three factors were root herbivores (RH hereafter),
FH and detritivores (D). FH are defined here as any species that feeds
on buds, flowers and fruits regardless of whether they also feed on
other parts of the plant. A total of 15 plants were assigned to each of
the treatments (five plants per block). One third-instar larva of the detritivore Morica hybrida Charpentier (Coleoptera: Tenebrionidae) was
added to the soil of each plant assigned to the D+ treatments. One
third-instar larva of the root-feeding herbivore Cebrio gypsicola Graells (Coleoptera: Cebrionidae) was added to the soil of each plant of
the RH+ treatments. M. hybrida and C. gypsicola are among the most
abundant generalist below-ground detritivores and herbivores,
respectively, to be found in the study area (see Doblas-Miranda, González-Megı́as & Sánchez-Piñero 2007 for more details on belowground fauna in the study area). The experiment was ended at the end
2010 The Authors. Journal compilation 2010 British Ecological Society, Journal of Animal Ecology, 79, 923–931
Above–belowground indirect interactions 925
of June (2007). The recovery rate of detritivores and RH at the end of
the experiment was very high (94Æ16% and 91Æ67% respectively).
Naturally occurring FH were allowed to lay eggs on all FH+
treatment plants, when these plants started to produce flowering
stalks. Three species of FH fed on the experimental plants during the
experiment, P. daplidice, E. crameri and P. xylostella. All plants
belonging to the FH+ treatments had at least one pierid larva
(2Æ46 ± 0Æ43 pierid eggs ⁄ plant) during the experiment, and 33Æ9% of
the plants had at least one P. xylostella larva (0Æ5 ± 0Æ11 Plutella
eggs ⁄ plant). For the FH) treatments, the experiment was checked
every 3 days using magnifying glasses and all eggs or larvae of FH
were removed by hand from plants.
DATA COLLECTION
Attack rate and abundance of above-ground herbivores
and parasitoids
To score free-living herbivores, the number of naturally occurring
sap-suckers (aphids and leafhoppers) and leaf herbivores was
recorded on each experimental plant every 3 days. Total abundance
of sap-suckers and leaf herbivores was calculated by summing the
number of individuals recorded during all the surveys until the end of
the experiment (end of June). To avoid problems of summing individuals counted in a previous census, we subtracted at each census the
number of individuals of the same instar ⁄ type (winged vs. not
winged) counted in the previous census. The attack rate was calculated as the probability of a plant being attacked by sap-suckers and
leaf herbivores (presence ⁄ absence) respectively.
To score seed predators, fruits were collected after complete maturation of seeds but before seed dispersal (28 June 2007). Seed predators were not considered FH because the feed only on mature seeds
not altering any other reproductive part of the plant. The abundance
and attack rate (proportion of fruits infested by seed predators) were
quantified in the lab by opening 10 fruits per plant of larger plants
and all fruits of plants which had produced <10 fruits. The abundance of seed predators was quantified by counting the total number
of fruits with at least one predator. Because it is possible to find up to
three seed predators per fruit, this is a conservative estimate of seedpredator abundance (see Gómez & González-Megı́as 2002, for a similar procedure).
Cotesia kazak Telenga (Braconidae, Hymenoptera) was the only
parasitoid species that attacked P. daplidice and E. crameri in the
study area during the study period. Infected larvae of both species
are easily identified because of the change in colour and appearance.
The solitary parasitoid builds its cocoon on the stems of M. moricandioides. We recorded the proportion of larvae attacked by
parasitoids per plant (attack rate), and the abundance of parasitoids
per plant as the total number of cocoons found until the end of the
experiment.
INDUCTION OF CHEMICAL DEFENCES
To measure GS concentrations of the above-ground plant tissue, the
youngest leaf of one stem of each of the experimental plants was collected at the end of the experiment (28 June 2007). Leaf samples were
immediately stored in 100% methanol to avoid any degradation by
myrosinase activity. Samples were freeze-dried, and the dried material was ground and extracted three times in 80% methanol after the
addition of p-hydroxybenzyl GS as an internal standard. Supernatants of extracts were applied to DEAE Sephadex A-25 (SigmaAldrich, St. Louis, MO, USA) columns (0Æ1 g in 2 mL of 0Æ5 m acetic
acid buffer, pH 5, per column, incubated overnight). Columns were
washed several times with water followed by two washes with 0Æ02 m
acetic acid buffer, pH 5. Purified sulfatase of Helix pomatia (EC
3.1.6.1, type H-1; Sigma, Taufkirchen, Germany), dissolved in 0Æ02 m
acetic acid buffer, was applied on each column and GS converted to
desulfoglucosinolate overnight. Desulfoglucosinolate were eluted
from the columns with 6 · 1 mL of water, and the samples were analysed by HPLC (1200 Series; Agilent Technologies, Inc., Santa Clara,
CA, USA) coupled with a diode-array detector on a Supelcosil LC18 column (250 · 4Æ6 mm, 5 lm; Supelco, Bellefonte, PA, USA)
using a gradient from water to methanol. Desulfoglucosinolates were
identified by comparison of UV-spectra and retention-times to those
identified in earlier studies (Müller & Sieling 2006). Response factors
of 0Æ5 for p-hydroxybenzyl GS, 1 for aliphatic and 0Æ26 for indolic GS
were considered.
STATISTICAL ANALYSES
Generalized linear models were used to test the effects of each factor
(FH, RH and D) and their interactions on herbivores (attack rate
and herbivore abundance) and on GS concentrations. Block was
included in all the analyses to control for the potential effect of the
location. Herbivore attack rates were fitted to a binomial distribution
with logit as link function, GS concentrations to a normal distribution with identity as link function, and herbivore abundance to a
Poisson distribution with log as link function.
Similarly, we used generalized linear models to test the effects of
RH and D factors on the parasitoid attack rate and abundance. Parasitoid attack rate was fitted to a binomial distribution with logit as
link function, whereas parasitoid abundance was fitted to a Poisson
distribution with log as link function.
Generalized linear models were also used to determine the relationship between GS (the explanatory variable) and insect abundances
(the response variable). Herbivore abundances were fitted to a Poisson distribution with log as link function in all analyses. The FH
effect on the induction of some GS was modified in some of the analysis by D+; therefore, we tested the relationship between those specific GS and herbivore abundance for: (i) all data; (ii) in the presence
of D (D+); and (iii) in the absence of D (D)). Mean ± SE is shown
throughout the manuscript.
Results
INSECT COMMUNITY ASSOCIATED WITH M.
MORICANDIOIDES
More than 62% of the plants were infected during the study
period with different species of aphids. The generalist Myzus
persicae Sulzer was the most abundant aphid species (92Æ8%
of total abundance), followed by the specialist Lipaphis erysimi Kaltenbach (5Æ3%) and other unidentified species
(1Æ9%). In addition, 80% of the plants were also infected by
the generalist planthopper species Agalmatium bilobum Fieber (Hemiptera, Issidae).
Foliar herbivores also attacked the experimental plants,
colonizing around 20% of them. The most abundant species
found were: larvae of P. rapae (5%; specialist on Brassicaceae), the moth Spodoptera sp. (Lepidoptera, Noctuidae;
48Æ3%) and the specialist sawfly Tenthredo sebastiani Lacourt
(Hymenoptera, Tenthredinidae; 46Æ7%).
2010 The Authors. Journal compilation 2010 British Ecological Society, Journal of Animal Ecology, 79, 923–931
926 A. González-Megı´as & C. Müller
Table 1. Results of the generalized linear models (chi-square values) showing the effect of the different factors (FH, RH and D; see Materials
and methods) on insect abundance (aphids, planthoppers, foliar herbivores and seed predators), and three glucosinolate (GS) concentrations
Whole model
FH
RH
D
FH · RH
FH · D
RH · D
FH · RH · D
Block
d.f.
Planthoppers
Foliar
herbivore
Seed
predators
Aphids
2-S-2-Hydroxy3-butenyl GS
4-Hydroxyindol
-3-ylmethyl GS
Indol-3-ylmethyl
GS
9.97
1
1
1
1
1
1
1
2
212Æ88***
34Æ65***
1Æ85
2Æ90**
1Æ73
1Æ96
8Æ07**
0Æ03
121Æ49***
19Æ65*
4Æ88*
0Æ94
6Æ24*
1Æ41
0Æ86
4Æ75*
3Æ31
5Æ57
40Æ02***
4Æ01*
1Æ51
8Æ03**
4Æ13*
3Æ89
0Æ06
0Æ46
20Æ19***
1254Æ50***
115Æ96***
0Æ37
157Æ72***
185Æ14***
0Æ18
4Æ05*
124Æ37***
707Æ99***
22Æ94**
0Æ01
6Æ03*
1Æ07
2Æ50
4Æ99*
1Æ31
1Æ09
8Æ35*
18Æ05*
3Æ62ms
0Æ64
4Æ78*
0Æ11
6Æ75**
0Æ46
0Æ41
4Æ73
23Æ17**
1Æ21
8Æ27**
0Æ57
0Æ0001
2Æ63
0Æ43
1Æ17
10Æ88**
FH, floral herbivores; RH, root herbivores; D, detritivores.
Chi-square values: msP = 0Æ07; *P < 0Æ05; **P < 0Æ01; ***P < 0Æ001.
More than 50% of the plants had at least one fruit attacked
by a seed predator. The only species found on M. moricandioides fruits until now in the study area was an unidentified
moth species of the family Blastobasidae (Lepidoptera).
Although the model for the abundance of parasitoids was
not significant (Appendix S1), there was a significantly positive effect of D presence on parasitoid abundance (0Æ23 ±
0Æ01 parasitoids ⁄ plants on D) vs. 0. 46 ± 0Æ11 on D+).
ATTACK RATE AND ABUNDANCE OF ABOVE-GROUND
GLUCOSINOLATE CONCENTRATIONS
HERBIVORES
Above-ground herbivore attack rates were not affected by
our experiment (P > 0Æ05, in all analyses). However, there
was a significant effect on aphid, planthopper, foliar herbivore and seed-predator abundances (Table 1).
Planthopper abundance was negatively affected by FH
(Table 1; Fig. 1a). The two-way interaction between RH and
D was significant (Table 1). Planthoppers were negatively
affected by D but only in RH) treatments (Fig. 1a).
Foliar herbivore abundance was positively affected by FH
(Fig. 1b). The significant interaction term between RH and
D indicated that foliar herbivores abundance was negatively
affected by D and RH, and only in RH)FH) treatment their
abundance was significantly higher than in the other treatments (Fig. 1b).
Seed-predator abundance was negatively affected by D
(Table 1; Fig. 1c). There was also a significant interaction
between RH and FH (Table 1). Only in RH) treatments
there was a clear negative effect of FH on seed predators
(Fig. 1c).
There was a significant treatment effect on aphid abundance (Table 1), however, the significant three-way interaction term indicated no main effect of any of the factors
(Table 1). FH-and D-affected aphid abundance on plants
but, in both cases, their effects were modified by the presence
of RH. Aphid abundance was negatively affected by FH and
D but only when RH was present (Fig. 2).
ATTACK RATE AND ABUNDANCE OF PARASITOIDS
Parasitoid attack rate differed between treatments because of
D (P < 0Æ05; Appendix S1). Attack rate was higher in D+
treatments (0Æ11 ± 0Æ05 D) vs. 0Æ35 ± 0Æ08 D+; Appendix
S1).
Seven GS were found in M. moricandioides leaves, four aliphatic and three indolic GS. Total GS concentrations were
on average 7Æ31 ± 0Æ46 lmol g)1 dry weight. The variation
of all GS was rather high but only the concentration of 2-S-2hydroxy-3-butenyl GS, 4-hydroxyindol-3-ylmethyl GS and
4-hydroxyindol-3-ylmethyl GS on M. moricandioides varied
significantly between treatments (Table 1).
RH+ negatively affected the concentration of 2-S-2hydroxy-3-butenyl GS in plants (Table 1; Fig. 3). Plants from
FH+ treatments also had lower concentrations of 2-S-2hydroxy-3-butenyl GS, but only in the presence of D (Fig. 3)
as indicated by the significant interaction term (Table 1).
The concentration of 4-hydroxyindol-3-ylmethyl GS differed significantly between treatments (Table 1). As shown
by the interaction term, FH+ plants had lower 4-hydroxyindol-3-ylmethyl GS concentrations but only in the absence of
D (Fig. 3).
The concentration of indol-3-ylmethyl GS also varied
between treatments (Table 1; Fig. 3). In this case, RH
induced the production of indol-3-ylmethyl GS in leaf tissue
(Fig 3).
RELATIONSHIPS BETWEEN GLUCOSINOLATES AND
INSECT ATTACK RATE AND ABUNDANCE
Aphid abundance was positively related to 2-S-2-hydroxy-3butenyl GS (b = 0Æ72 ± 0Æ32; Appendix S2), but the interaction was significant only in D+ (b = 4Æ06 ± 0Æ56; Appendix S2). Foliar herbivore abundance was also positively
related to 2-S-2-hydroxy-3-butenyl GS (b = 11Æ09 ± 1Æ61;
Appendix S2), the relationship was not affected by D
(P > 0Æ05). The relationship was significantly negative for
seed predators (b = )2Æ96 ± 1Æ03), but it was modified by
D (Appendix S2). There was no significant relationship for
2010 The Authors. Journal compilation 2010 British Ecological Society, Journal of Animal Ecology, 79, 923–931
Above–belowground indirect interactions 927
(a) 7
8
5
N planthoppers
N planthoppers
6
*
4
3
2
a
a
a
6
b
4
2
1
0
FH–
0
FH+
D–
D+
D–
RH–
(b) 0·5
1·0
0·8
N folivores
N folivores
0·4
*
0·3
0·2
a
0·6
b
0·4
b,c
c
0·2
0·1
0·0
0
FH–
FH+
D–
D+
D–
RH–
(c)
1·5
0·5
D–
N aphids
30
18
12
6
b
b
b
b
b
c
3·0
a
b
b
2·0
b
1·0
0·0
0
a
24
N seed predators
N seed predators
*
2·0
42
36
D+
RH+
4·0
2·5
Fig. 1. Effects of flower herbivores (FH),
root herbivores (RH) and detritivores (D)
on : (a) planthopper abundance (N planthoppers), (b) foliar herbivore abundance
(N folivores) and (d) seed predator abundance (N seed predators). *Significantly different (P < 0Æ05). Treatments with different
letters are significantly different (post hoc
log-likelihood ratio test, P < 0Æ05).
D+
RH+
c
0
FH–RH–D–
FH–RH+D–
FH–RH+D+
FH+RH+D–
FH–RH–D+
FH+RH–D–
FH+RH–D+ FH+RH+D+
Fig. 2. Effects of flower herbivores (FH), root herbivores (RH) and
detritivores (D) on aphid abundance (N aphids). Treatments with
different letters are significantly different (post hoc log-likelihood
ratio test, P < 0Æ05).
planthoppers. Furthermore, there was a significantly negative relationship between parasitoid attack rate and 2-S-2hydroxy-3-butenyl GS (b = )26Æ02 ± 12Æ03,) but only in
D+ (Appendix S2). This interaction was also marginally significant for parasitoid abundance in the presence of D
(b = )9Æ91 ± 6Æ90; *Appendix S2).
Furthermore, there was a significantly negative relationship between 4-hydroxyindol-3-ylmethyl GS and aphid abundance (b = )8Æ56 ± 1Æ29), and seed predator abundance
(b = )24Æ75 ± 7Æ41; Appendix S2). Planthopper abundance
was positively correlated with this GS (b = 3Æ01 ± 0Æ37;
Appendix S2). The relationship was not significant for foliar
D+
FH–
FH+
RH–
FH–
FH+
RH+
herbivores or parasitoids. D did not modify any of the interactions between insect abundance and this GS.
With regard to indolic GS, the abundances of aphids and
planthoppers (b = )1Æ90 ± 0Æ26 and )1Æ94 ± 0Æ43 respectively), as well as seed predators (b = )2Æ84 ± 0Æ79; Appendix S2) were negatively related to indol-3-ylmethyl GS
concentrations. There was no significant relationship
between GS concentrations and other insects.
Discussion
ABOVE- AND BELOW-GROUND HERBIVORE EFFECTS ON
ABOVE-GROUND ORGANISMS
Our experiment demonstrates that both above- and belowground herbivores significantly affect other herbivores
co-occurring on M. moricandioides (Fig. 4). Antagonistic
relationships between herbivores are usually expected but
also positive effects between herbivores feeding on the same
plant have been reported in previous studies (Agrawal &
Sherriffs 2001; Hopkins et al. 2009). In most cases, the underlying mechanism seems to be related to the induction of plant
metabolites. Feeding damage can induce the production of
secondary compounds by the plant, attracting specialist herbivores, which in turn trigger a boost in the plant’s chemical
2010 The Authors. Journal compilation 2010 British Ecological Society, Journal of Animal Ecology, 79, 923–931
928 A. González-Megı´as & C. Müller
(a)
0·10
a
a
0·10
0·08
4-hydroxyindol GS
2-S-2-hydroxy GS
0·12
b
b
0·06
0·04
0·02
a
0·06
0·04
b
b
b
0·02
0·00
0·00
FH–
(b) 0·10
FH+
D–
FH–
FH+
D+
FH–
FH+
D–
FH–
FH+
D+
0·12
Indol-3-ylmethyl GS
2-S-2-hydroxy GS
0·08
0·08
*
0·06
0·04
0·02
0·00
0·10
*
0·08
0·06
0·04
0·02
0·00
RH–
RH+
RH–
(a)
Parasitoids
+
Seed predators
–
–
Aphids
–
Floral herbivores
+
+
Planthoppers
Foliar herbivores
–
–
–
Detritivores
Root herbivores
(b)
–
Parasitoids
+
Seed predators
–
Floral herbivores
Aphids
–
+
+
Planthoppers
Foliar herbivores
–
–
Detritivores
Fig. 4. Effects of floral herbivores and decomposers on other organisms associated with Moricandia moricandioides (a) in absence of root
herbivores and (b) in presence of root herbivores.
RH+
Fig. 3. Effects of flower herbivores (FH),
root herbivores (RH) and decomposers (D)
on concentrations (lmol g)1 dry weight) of
2-S-2-hydroxy-3-butenyl GS (2-S-2-hydroxy
GS), 4-hydroxyindol-3-ylmethyl GS (4hydroxyindol GS) and indol-3-ylmethyl
GS. *Significantly different (P < 0Æ05).
Treatments with different letters are significantly different (post hoc log-likelihood ratio
test, P < 0Æ05).
defences (but see Hopkins et al. 2009 for a review). In our
system, FH favoured the abundance of foliar herbivores on
M. moricandioides. Although FH had a strong effect on the
GS content in M. moricandioides (Fig. 5), individual GS
responded in different ways, being either reduced or
increased. Thus no general pattern could be derived from our
experiment. Because FH also had a negative effect on aboveground plant biomass and no effect on the quality of the
above-ground tissue in terms of N content (A. GonzálezMegı́as, unpublished) none of those factors seem to clarify
the positive effect of FH on foliar herbivores.
Most interactions between FH and RH with the other
co-occurring herbivores were negative and were mediated by
the plant (Figs 4 and 5). FH had a negative effect on aphid
and plant-hopper abundance. Similar negative effects of
chewing herbivores on sap-suckers have been reported in
several studies (Inbar et al. 1999; González-Megı́as & Gómez
2003; van Zandt & Agrawal 2004; Poveda et al. 2005; Gómez
& González-Megı́as 2007). Two main mechanisms have
been proposed for these negative effects on sap-suckers feeding on stems associated with the reproductive parts of the
plant such as flower stalks, buds and even flowers: induced
resistance by altered plant chemical defences (Faeth 1992;
Faeth & Wilson1997; Denno & Kaplan 2007), and reduced
resources (see Poveda et al. 2005). In the M. moricandioides
system, FH led to reduced concentrations of GS, specifically
2-S-2-hydroxy-3-butenyl GS and 4-hydroxyindol-3-ylmethyl
GS. Conversely, aphid abundance was positively correlated
with 2-S-2-hydroxy-3-butenyl GS. Whether this is because of
a preference of the aphid species for this GS, or whether the
aphids induced particularly 2-S-2-hydroxy-3-butenyl GS, or
both, can not be discriminated. It has been shown that
aphids, at least specialist species, prefer tissue with higher GS
concentrations (Gabrys, Tjallingii & van Beek 1997), allowing them to escape from competition (Hopkins et al. 2009).
However, the most abundant aphid species on M. moricandioides was a generalist aphid, M. persicae. In addition, the
2010 The Authors. Journal compilation 2010 British Ecological Society, Journal of Animal Ecology, 79, 923–931
Above–belowground indirect interactions 929
(a)
Parasitoids
Seed predators
–
Floral herbivores
indol-3
–
–
–
Aphids
+
–
2-S-2Hydroxy
+
Planthoppers
Foliar herbivores
+
–
Root herbivores
Detritivores
(b)
Seed predators
–
–
indol
–
–
Parasitoids
–
Floral herbivores
4Hydroxy
–
–
Aphids
+
Planthoppers
2-S-2Hydroxy
DETRITIVORE EFFECTS ON ABOVE-GROUND
+
ORGANISMS
Foliar herbivores
+
Root herbivores
Milbrath & Nechols 2004). An additional mechanism
observed in other systems is incidental predation. FH can
incidentally predate on seed predator eggs when feeding on
flowers (Gómez & González-Megı́as 2002, 2007). In addition,
FH affected seed-predator abundance by inducing changes
in GS concentrations (TMII). In this case, the negative interaction between FH and seed predators was somewhat mitigated by the negative effect of FH on the production of
aliphatic GS, which were negatively related to seed predator
abundance. The RH effect on seed predators was also related
to GS induction. There was a clear negative relationship
between seed predators and indol-3-ylmethyl GS, which was
induced in the study system by RH. By inducing this GS or
through changes of plant emitted volatile pattern, RH probably increased host-plant attractiveness to parasitoids of different herbivores, as found in other studies (Masters, Jones &
Rogers 2001; van Dam et al. 2003).
Herbivore interactions on M. moricandioides are therefore
mainly based on indirect interactions: on density-mediated
indirect interactions by reducing resources, and on TMII
through altered concentrations of chemical plant compounds, i.e. GS or other traits. The response of the plant to a
given herbivore species as well as the response of herbivores
to the plant chemical profile is highly species-specific with
regard to the induction and effect of individual GS.
–
Fig. 5. Effects of floral herbivores and root herbivores on three
different glucosinolates (GS) (indol-3, indol-3-ylmethyl GS;
4-hydroxy, 4-hydroxyindol-3-ylmethyl GS; 2-S2-hydroxy, 2-S-2hydroxy-3-butenyl GS), and the relationship between GS and the
other insects associated to the host-plant (a) in presence of detritivores and (b) in absence of detritivores. Dashed line, effects on GS;
solid line, responses to GS (or other unidentified correlated traits).
reduction of resources (a density-mediated indirect interaction) can be an alternative mechanism by which
FH-affected sap-suckers because FH reduced the production
of flower stalks and flower production in M. moricandioides
(A. González-Megı́as, unpublished) and thereby diminished
the resources for sap-suckers.
Floral herbivores and RH also negatively affected seed
predators (Fig. 4). FH might affect seed predators by exploitative competition, as a consequence of reducing the availability of oviposition sites. FH reduced flower production
by nearly 50% in M. moricandioides during the study period
(A. González-Megı́as, unpublished). Similar negative effects
of FH on seed-predator abundance have been observed in
other systems (Juenger & Bergelson 1998; Gómez & González-Megı́as 2002, 2007; Freeman, Brody & Neefus 2003;
Detritivore effects on plant growth and reproductive success
can lead to indirect interactions with above- and belowground organisms sharing the same host plant, as found in
several studies (Bonkowski et al. 2001; Scheu 2001; Bezemer
& van Dam 2005; Bezemer et al. 2005; Wolfe et al. 2005;
Kessler & Halitschke 2007; Rasmann & Turlings 2007).
Some of these studies found a positive effect of detritivores in
sap-suckers (Bonkowski et al. 2001; Poveda et al. 2005),
which was apparently associated with changes in the content
of soluble nitrogen or amino acids in plant phloem (Bonkowski et al. 2001). However, we found a negative effect of detritivores on the proportion of N content on above-ground
tissue (A. González-Megı́as, unpublished). This negative
effect of D on plant quality could explain the negative effects
of D on aphid, plant-hopper, seed predator and leaf
herbivore abundance in the M. moricandioides system.
Although not directly related to roots, detritivores can
alter plant defence levels such as GS concentrations (Bezemer
& van Dam 2005; Lohmann et al. 2009) and thereby indirectly affect herbivores. The effects appear to be related to
changes in nitrogen availability to the plants as well as to
changes in gene expression related to plant defence (Bezemer
& van Dam 2005). In Sinapis alba L., aromatic GS concentrations significantly increased because of the presence of earthworms (Lohmann et al. 2009). In M. moricandioides, D
presence altered the effects of FH in the production of aliphatic GS, supporting the hypothesis that D prompts
changes in plant chemistry (Fig. 5).
2010 The Authors. Journal compilation 2010 British Ecological Society, Journal of Animal Ecology, 79, 923–931
930 A. González-Megı´as & C. Müller
It bears noting that D presence also affected parasitoid
abundance on M. moricandioides, although the mechanism
involved is not clear. The use of induced volatile metabolites
to attract natural enemies is a mechanism observed in other
systems for both specialists and generalist parasitoids (Turlings et al. 2002; Soler et al. 2005). Isothiocyanates and nitriles, the hydrolysis products of GS, are known to be involved
in attraction of parasitoid species in other Brassicaceae systems (Bradburn & Mithen 2000; Mumm et al. 2008) and may
have been involved also in parasitoid attraction in the M. moricandioides system. However, an increased attraction of
plants to parasitoids after detritivore activity is a misleading
signal to the parasitoids and should therefore not be evolutionary stable.
system remain unknown and more specific and mechanistic
experiments are necessary to elucidate this issue. Jasmonic
acid may be an important signal mediating the modifications
of plant metabolites such as GS, as shown earlier (Textor &
Gershenzon 2009) but also the release of volatile organic
compounds (Ozawa et al. 2000), which attract herbivores
and parasitoids from a distance.
Summarizing this study shows that below-ground organisms through plant-mediated indirect effects can shape the
interactions among above-ground organisms. One of the
most important and novel result is that detritivores induced
changes in plant metabolites, varying the quality and attractiveness of plants to herbivores and parasitoids.
Acknowledgements
COMPLEX INTERACTIONS AMONG FOOD WEB
COMPONENTS
More than 50% of the pairwise interactions of FH, RH and
D with the co-occurring organisms of the community and
with the plant GS were not additive but were affected by
another manipulated component. These interactions did not
affect the manipulated organisms with the same intensity, as
most D effects were influenced by RH, some FH effects were
influenced by RH or D, but barely any of the RH effects
appeared to have been influenced by any of the other organisms (Figs 4 and 5). Indeed, two different scenarios of the
multiple trophic relationships in our system arose, depending
on the presence of RH (Fig. 4). Most of the negative relationships between FH and D on the other herbivores were evident
only in the absence of RH. The impacts of RH on plants are
well-known, ranging from effects on plant chemical defences
to the modification of the phloem nitrogen concentration,
which can increase sap-sucker abundance (Gange & Brown
1989; Masters & Brown 1997; Masters et al. 2001; Poveda
et al. 2005). Therefore, RH probably had an indirect negative
effect on aphids via induced defences but at the same time a
positive interaction with aphids via other modifications of
plant traits. This positive interaction offset the negative effect
of FH and D on aphids, acting as a buffer. Even more importantly, the influence of FH on GS concentration was clearly
altered by detritivores.
Surprisingly, two alternative scenarios also become
evident in our system with regard to plant chemical defences
(Fig. 5), where plant secondary metabolites and the relationship with other co-occurring organisms varied according to
the presence of detritivores (Fig. 5). Detritivores altered the
influence of FH on GS, potentially affecting the response of
the other insects to the plant defence mechanisms. It is important to highlight that most of these complex interactions
occurred between organisms belonging not only to the same
trophic guild but also to the same food web compartment,
thereby increasing the complexity of the food web functioning and dynamics. In a less complex experiment, where not
all of the interacting organisms are considered, such interactions would remain unnoticed. Many of the mechanisms by
which a third species altered a pair-wise interaction in our
The authors would like to thanks Francisco Sánchez-Piñero, and José Antonio
Hódar for their frequent discussion on food web interactions, and their help
in the field. We thank J. M. Nieto Nafrı́a (Aphids), T. Oltra and J. Vicente
Falco (Braconidae), Oscar Aguado (Tenthedinidae) and Vladimir Gnezdilov
(Issidae) for the identification of the specimens. Jose M. Gómez, Michael
Rostás, Martı́n Pareja and Rosa Menendez kindly revised an early version
of this manuscript. This work was partially funded by CICYT grant
BOS2001-3806.
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Received 12 October 2009; accepted 15 February 2010
Editor: Karl Cottenie
Supporting Information
Additional Supporting Information may be found in the online
version of this article.
Appendix S1. Results of the generalized linear models showing the
effects of root herbivores (RH) and decomposers (D) on pierid
parasitoids.
Appendix S2. Models of generalized linear models for insect abundances and glucosinolate concentration on M. moricandiodes.
As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials may be
re-organized for online delivery, but are not copy-edited or typeset.
Technical support issues arising from supporting information (other
than missing files) should be addressed to the authors.
2010 The Authors. Journal compilation 2010 British Ecological Society, Journal of Animal Ecology, 79, 923–931