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Negative effects of vertebrate on invertebrate herbivores mediated by enhanced

plant nitrogen content

Article  in  Journal of Ecology · November 2018

DOI: 10.1111/1365-2745.13100

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 Journal of Ecology
Accepted Article
DR JORDI F. PAGÈS (Orcid ID : 0000-0001-9346-8312)

PROFESSOR DELI WANG (Orcid ID : 0000-0001-6576-9193)

DR ZHU HUI (Orcid ID : 0000-0003-0348-5560)

DR LING WANG (Orcid ID : 0000-0002-9916-2346)

Article type : Research Article

Handling Editor: Samantha Chapman

Running Head: Indirect interactions between herbivores

Negative effects of vertebrate on invertebrate herbivores mediated by enhanced plant nitrogen

content

YU ZHU12, ZHIWEI ZHONG1, JORDI F. PAGÈS3, DEBORAH FINKE4, DELI WANG1, QUANHUI MA1, NAZIM HASSAN1, HUI
ZHU 1, LING WANG1

1
Institute of Grassland Science and School of Environment, Northeast Normal University, and Key
Laboratory of Vegetation Ecology, Ministry of Education, Changchun, Jilin 130024 China

2
Departments of Aquatic Ecology, Netherlands Institute of Ecology (NIOO-KNAW), Wageningen 6708
PB The Netherlands

This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/1365-2745.13100

This article is protected by copyright. All rights reserved.

 3
School of Ocean Sciences, Bangor University, Menai Bridge, Isle of Anglesey LL595AB United
Kingdom
Accepted Article
4
Division of Plant Sciences, University of Missouri, Columbia MO 65211 USA

Corresponding author: DELI WANG

Tel: 86-431-85099737; Fax: 86-431-85695065

Email: wangd@nenu.edu.cn

Abstract

1. Classic theory holds that the main interaction within the herbivore guild is competition, based
on research focused on co-occurring, similarly-sized species that reduce the quantity of shared plant
resources. However, plant quality may also be crucial in mediating herbivore interspecific
interactions. This is especially true when competition occurs between distantly-related herbivore
species, given that small terrestrial herbivores (e.g. insect herbivores) appear to be more sensitive to
alterations of plant quality than plant quantity.

2. In this study, we first tested in the field whether large vertebrate herbivores (cattle Bos taurus)
exerted a negative effect on smaller insect herbivores (grasshopper Euchorthippus unicolor) through
their overlapping foraging preferences for a dominant grass Leymus chinensis. We measured
changes in grass quantity, grass quality, and microclimatic conditions in response to vertebrate
grazing and conducted additional manipulative studies in the field and the laboratory to identify
potential mechanisms underlying the interaction.

3. Our results showed that grazing by large herbivores caused a significant decline in grasshopper
population density and individual performance (survival, size and weight of both female and male E.
unicolor), despite a 38% increase in grass nitrogen (N) content in grazed plots. Experiments
manipulating N levels of L. chinensis in the field and the laboratory confirmed that enriching plant N
had a negative effect on grasshopper individual performance and population size. Therefore,
enhanced quality (N content) of plant resources appears to be an important driver in mediating the
negative effect of vertebrate grazing on grasshoppers.

4. Synthesis. We document that phylogenetic relatedness and trait similarity can be poor
predictors of interaction strength in some cases, since distantly related herbivores of disparate size
can interact indirectly via changes in plant quality. Counter-intuitively, the observed negative effect

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 of cattle on grasshoppers was mediated, at least in part, by an increase in plant quality in cattle
grazed areas. The implication is that light to moderate grazing, a common management strategy,
Accepted Article
may contribute to suppression of grasshoppers in the Eurasian steppe grassland system by altering
plant nutrient supplies.

Key words: plant quality; indirect interactions; large herbivores; nutrient requirement; species
coexistence; body size; density-mediated indirect effect; trait-mediated indirect effect.

INTRODUCTION

Competition theory predicts that ecological similarity among species enhances the intensity of their
interactions (Connell, 1983; Schoener, 1974). Thus, in the case of interactions among species within
the herbivorous guild, the vast majority of work has traditionally focused on closely related species
of similar size (e.g., Denno et al., 2000; Karban, Grof-Tisza, & Holyoak, 2012; Odadi, Karachi,
Abdulrazak, & Young, 2011). However, competition among herbivores does not always conform to
traditional paradigms, and phylogenetic relatedness and trait similarity can be poor predictors of
interaction strength in some cases (Eubanks & Finke, 2014; Gurevitch, Morrow, Wallace, & Walsh,
1992; Hochberg & Lawton, 1990; Kaplan & Denno, 2007). Increasingly, ecologists are recognizing the
fact that interactions among distantly related herbivores are prevalent and relevant, with
far-reaching effects on community assembly, organization and dynamics (Bakker, Dobrescu, Straile,
& Holmgren, 2013; Belovsky, 1986; Pringle, Young, Rubenstein, & McCauley, 2007).

Competition for shared host plants between large grazing mammalian herbivores and smaller
herbivores, such as arthropods, is a classic example of interaction among phylogenetically distinct
groups. Recent meta-analytical reviews of the literature find that, on average, the effects of large
herbivores on smaller animal taxa are negative, both in terms of abundance and diversity (Foster,
Barton, & Lindenmayer, 2014; Takagi & Miyashita, 2014; van Klink, van der Plas, van Noordwijk,
WallisDeVries, & Olff, 2015); however, there is considerable variation in the outcome of individual
studies including positive (Cao et al., 2015), negative (Pringle et al., 2007), and neutral effects (Riipi,
Lempa, Haukioja, Ossipov, & Pihlaja, 2005) of large herbivores on insect populations. While some of
this context-dependency is attributed to differences in the spatial and temporal scale of the study,
differences across systems in the processes linking large and small herbivores also play a significant
role (Takagi & Miyashita, 2014).

The mechanisms underlying the effects of large herbivores on smaller herbivores include both
direct and indirect pathways (as reviewed by van Klink et al., 2015). Direct interactions result from
incidental ingestion or trampling of smaller herbivores by large herbivores (Gómez &
González-Megías, 2002). Indirect effects may result from changes in plant quantity (i.e.,

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 density-mediated indirect effect), such as when large herbivores reduce plant biomass (Huntzinger,
Karban, & Cushman, 2008; Vandegehuchte, Schütz, de Schaetzen, & Risch, 2017), thus limiting the
Accepted Article
availability of resources for other herbivores. The outcome of competition between differently-sized
herbivores tends to be highly asymmetrical, thus these indirect effects are typically negative for the
abundance of the smaller herbivore and neutral for the larger (Gómez & González-Megías, 2002).
However, in situations where the smaller herbivore prefers a different host plant than that
consumed by the large herbivore, facilitation (i.e. commensalism/mutualism) may occur. As an
example, Zhong et al. (2014) found that domestic sheep benefit grasshoppers by selectively
consuming Artemisia forbs. The reduction in forb biomass enhances the ability of grasshoppers to
locate their preferred Leymus grass host plant. Indirect effects may also result from
herbivore-induced changes in host plant quality or architecture (i.e., trait-mediated indirect effect)
(e.g., Ohgushi, 2005). For example, in the African savanna large grazers are reported to benefit other
grazers (McNaughton, 1976), either by stimulating re-growth of plants, which is usually of better
quality than mature leaves, or by returning nutrients in the form of faeces and urine (McNaughton,
1979). Feeding and trampling may also decrease the height and structural complexity of vegetation,
thus altering the physical environment encountered by smaller herbivores (Huntzinger et al., 2008;
Pagès et al., 2012; Pringle, 2008). Simplification of plant structure may expose herbivores to harsh
abiotic conditions or render them more vulnerable to predators (Suominen, Danell, & Bergström,
1999; van Klink et al., 2015), a type of indirect effect known as environment-mediated interaction
modification (Wootton, 1992).

As highlighted above, large and small herbivores might interact through a diversity of pathways,
which depend on the traits of the entire suite of herbivores, the traits of the plant community and
environmental factors, making the prediction of the direction and strength of interactions a complex
matter. For arthropods, in particular, responses are most often attributed to altered plant biomass
and structure rather than changes in plant quality (Kruess & Tscharntke, 2002; Lind et al., 2017;
Takagi & Miyashita, 2014), but the impacts of grazer-induced changes in plant quality are often not
investigated directly (Lind et al., 2017; Moran, 2014). Indeed, alteration of plant quality is likely to
have profound impacts on small herbivores, especially for some specialist insects (Behmer & Joern,
2008; Belovsky, 1997). Current paradigms usually assume that insect herbivores in terrestrial
systems are N limited due to the relatively lower N content of plant tissues (Mattson, 1980). Most N
in plants is in the form of protein; therefore, an increase in plant N content can result in higher
herbivore performance by relieving constraints on growth and reproduction (White, 1993).
However, studies with grasshoppers (Cease et al., 2012; Clissold, Sanson, & Read, 2006) and
butterflies (Fischer & Fiedler, 2000; Tao, Berns, & Hunter, 2014) have demonstrated that ingesting
excessive N can lead to negative effects on insect performance by disrupting their nutritive
equilibrium (Raubenheimer & Simpson, 1993). Also, the enhancement of plant N could drive

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 detrimental effects on herbivorous insect performance by increasing the concentration of alkaloids
or other N-based allelochemicals (Behmer, 2009). In the latter case, high N content should not be
Accepted Article
equated to higher plant quality. Given the uncertainty in the response of insect herbivores to N, a
direct test of the effects of large mammalian herbivory on plant quality and its consequences for
phytophagous insects is needed.

Here, we explore the interaction between large domestic herbivores and small herbivorous
insects in a semiarid grassland ecosystem. The dominant plant community in this system is the grass
Leymus chinensis, with a variety of less abundant forb species (Wang, Du, Zhang, Ba, & Hodgkinson,
2017; Zhu et al., 2012). The herbivore community is dominated by the large vertebrate Bos taurus
(i.e. domestic cattle) and the small insect grasshopper Euchorthippus unicolor. Both cattle and E.
unicolor grasshoppers prefer L. chinensis grass, and rarely feed on forbs (Liu et al., 2015; Zhong et al.,
2014). Previous studies have found negative effects of ungulate grazing on the populations of
grass-feeding grasshoppers due to reductions in plant biomass (Capinera & Sechrist, 2012) and
positive effects of ungulate grazing due to increased structural heterogeneity (Joern, 2004), but did
not directly measure plant quality. One previous study specifically investigated the response of
grasshoppers to plant quality and found higher abundance of grasshoppers in heavily grazed areas
where grass N content was reduced, but did not measure the possibly confounding effects of grazing
on plant abundance and structure (Cease et al., 2012). No studies have directly manipulated the
presence of grazing herbivores and systematically examined multiple mechanistic pathways of
impact on grasshoppers, including altered plant quantity, microclimate conditions, and plant quality.

Our work is novel in that we used a set of field and laboratory manipulative experiments to
directly investigate the potential pathways whereby large domestic herbivores affect E. unicolor
grasshoppers, including plant quantity, microclimate conditions, and plant quality. We addressed the
questions: (i) does large herbivore grazing affect the individual performance and population
abundance of co-occurring E. unicolor grasshoppers, and if so, what is the underlying mechanism?
Specifically, we ask (ii) does large herbivore grazing modify plant resource availability (i.e.
density-mediated indirect effect)?, (iii) does large herbivore grazing modify the microclimatic
conditions experienced by small herbivores (i.e. environmentally-mediated indirect interaction)?,
and (iv) does large herbivore grazing modify plant resource quality (i.e. trait-mediated indirect
effect)?

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 MATERIALS AND METHODS
Accepted Article Study system and background

Our study was conducted at the Grassland Ecological Research Station, Northeast Normal University,
Jilin Province, China (44°45′ N, 123°45′ E). The study site is located in the eastern region of the
Eurasian steppe, where the climate is semiarid, with cold, arid winters and hot, rainy monsoonal
summers. Annual mean temperature ranges from 4.6 to 6.4 ◦C, and annual precipitation varies
between 280-400 mm, with 70% falling during the growing season, especially between June and
August.

The meadow steppe in the area is dominated by the perennial rhizomatous grass Leymus
chinensis, which has strong compensatory regrowth after grazing or clipping disturbance (Gao,
Wang, Ba, Bai, & Liu, 2008; Wang et al., 2017). L. chinensis provides food and substrate to a great
number and variety of native and domestic herbivores. Other common species at this site include
grasses such as Phragmites australis, Calamagrostis epigejos and Setaria viridis; and forbs such as
Artemisia scoparia and Messerschmidia sibirica (Liu et al., 2015). From the 1970s to 2011, the study
area was fenced to exclude livestock and human residents for grassland management and
conservation purposes.

The main large vertebrate herbivores in the area are domestic cattle (Bos taurus), which prefer
the dominant grass L. chinensis and rarely feed on forb species (Liu et al., 2015, also see Appendix
S1). The major invertebrate herbivores at the site are grasshoppers (Oedipodinae, Acrididae) and
leafhoppers (Hemiptera, Cicadellidae) (Zhong et al., 2017; Zhu et al., 2012). The Euchorthippus
grasshopper dominates the herbivorous insect community, accounting for > 65% of all insect
abundance annually (Zhong et al., 2017). Grasshopper E. unicolor usually displays one generation a
year and displays five nymphal stages: eggs typically hatch in late July to early August, becoming
adults by mid-August, and reaching peak densities by the end of August (Zhong et al., 2014). E.
unicolor shares similar dietary preferences with domestic cattle: it feeds predominately on L.
chinensis grass, and only seldom on forbs (Appendix S1). Thus, the diets of both dominant vertebrate
and invertebrate herbivorous species in this semiarid grassland ecosystem are completely
overlapping.

Domestic cattle were introduced in our study site in 2012, seasonally grazed from June through
September and their grazing is maintained at a light to moderate intensity (0.1-0.3 animal units/ha).
Before the onset of cattle grazing in May 2012, five 50 × 50 m fenced (1.5 m high) cattle exclosures

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 (ungrazed plots) were randomly established across the study site at 50 m intervals. Additional
unfenced 50 × 50 m plots served as controls (grazed plots) in areas adjacent to each of the five
Accepted Article
ungrazed plots, about 2 m apart. Control grazed plots were used to monitor the responses of plant
communities to cattle grazing.

Effects of cattle grazing on E. unicolor population density and performance

To investigate the effects of cattle grazing on E. unicolor density in grazed and ungrazed plots after 3
years of cattle grazing (question (i)), two parallel transects with a series of ten 0.25 m2 rings were
placed at about 6.5 m intervals and 10 m away from the plot boundary to minimize edge effects.
Rings were left undisturbed for at least one day before grasshopper surveys. Densities were
estimated by counting the number of E. unicolor in each ring (Joern, 2004). Surveys were conducted
on the 10th, 21st, 30th of August, and the 8th of September 2014. All surveys were conducted on sunny
days with minimal cloud cover and no wind. We averaged E. unicolor density for the two transects to
yield a single value for each plot (n = 5) on each of the 4 sampling days.

To assess the effects of cattle grazing on E. unicolor performance we determined survival rates,
body size and mass gain in the grazed and ungrazed plots. In August 2014, we installed 2 circular
cages (0.75 m2 basal area × 1.1 m high) in each plot (20 cages in total). Each circular cage was
constructed of iron rod frames, covered with 2-mm plastic mesh screen, and sunk 10 cm into the
ground. Just before the deployment of the circular cages, the area was cleared of all aboveground
invertebrates and predators using a vacuum cleaner. We then collected fourth-instar E. unicolor
using sweep-nets on the adjacent grassland area. Before the nymphs were introduced into the
cages, E. unicolor were separated according to sex, and their initial body mass was determined by
weighing seven individuals at a time in one vessel using an analytical scale (Top Instrument, Zhejiang,
China). E. unicolor were introduced into each circular cage in groups of 14 individuals (7 females and
7 males), which matched observed densities in the study site. Survival was estimated by counting
the number of E. unicolor per cage every three days, starting on the 8th of August and ending on the
28th of August. Adult body mass and size measurements were determined again for all the remaining
females and males from each circular cage. Mass gain was calculated by subtracting the initial mass
from the adult mass (Specht, Scherber, Unsicker, Köhler, & Weisser, 2008). We measured E. unicolor
body length and femur length using an Electronic Digital Caliper (Guanglu Inc., Guilin, China). The
average for the two circular cages within each plot was used in statistical analyses.

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 Effects of cattle grazing on vegetation abundance and microclimatic conditions
Accepted Article In order to test question (ii), i.e. whether cattle grazing influenced plant resource availability, we
examined the differences in plant communities in grazed and ungrazed plots. In August 2014, we
established two 50 m parallel transects within each plot and measured plant community
characteristics in ten 0.5 × 0.5 m quadrats along each transect, at about 4 m intervals. Within each
quadrat, we identified plant species, counted the number of shoots for each species (i.e. if the
species were clonal, this means we counted the number of ramets), visually estimated the
percentage cover of all plant groups, and measured plant height on 5 individual shoots for each
plant group (to the nearest centimetre) using a ruler. We estimated the volume (in m3) of three
different plant groups (L. chinensis, other grasses and forbs) by multiplying the mean area covered
with the mean height of each of the plant groups in twenty quadrats in each plot (Huntzinger et al.,
2008). We measured aboveground biomass by clipping standing plant material to 1 cm above
ground level from 8 randomly located 0.5 × 0.5 m quadrats within each plot. For each plant variable,
the values were averaged to yield a single value for each plot (the level of replication for this field
experiment, i.e. n=5).

In order to test question (iii), i.e. whether cattle grazing influenced within-canopy microclimatic
conditions, we evaluated light, temperature and humidity at 5 cm and 35 cm above ground
(grasshopper active zones) in August 2014, within the same quadrats used for vegetation variable
sampling. Photosynthetically active radiation (PAR) was measured on a clear day with a GLZ-C-G PAR
(photosynthetically active radiation) point sensor (Top Instrument, Zhejiang, China). Ambient air
temperature and relative humidity were measured using an AR-847 digital thermo-hygrometer
(Jinzhan Inc., Shenzhen, China).

Effects of cattle grazing on plant quality

To test question (iv), i.e. whether large grazers influenced plant quality, the chemical and physical
traits of the dominant grass L. chinensis were measured in grazed and ungrazed plots. We collected
L. chinensis leaves from 8 randomly located 0.5 × 0.5 m quadrats within each plot, in mid-August in
2014. Two of the upper-most (2nd or 3rd leaves from the top) fully expanded leaves that had not been
damaged by cattle or grasshoppers were measured. These tend to be the leaves that are most
commonly used by E. unicolor (Y. Zhu, field observations). Leaves were fresh- and dry-weighed to
determine water content. Then, dried samples were ground using a ball-mill and analysed for
nitrogen (N) and carbon (C) contents, and C:N ratio using a Vario MICRO cube Elemental Analyzer
(Elementar GmbH., Hanau, Germany). Leaf toughness of L. chinensis was measured on 10 individual

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 leaves in each plot using an improved SY-S03 penetrometer (Shiya Inc., Shijiazhuang, China). All
values were averaged to yield a single value for each plot (resulting in 5 replicates, n=5).
Accepted Article
Effects of nitrogen addition on E. unicolor grasshopper and L. chinensis grass

Our results in 2014 suggested that the negative influences of cattle on E. unicolor grasshoppers were
not a result of reduced availability of shared resources, nor a consequence of a change in
microclimatic conditions, but a result of alterations of plant quality (N content) (see Fig. 1-4 and
tables in the Results section below). Therefore, in 2015, we conducted a set of field and laboratory
fertilisation experiments to investigate how increased N content in plants affected E. unicolor
grasshoppers.

In April 2015, we set up eighteen 4 × 4 m plots that were randomly allocated to three levels of N
enrichment (0, 10 and 17.5 g N/m2, n = 6). N levels were chosen to produce an increase in plant N
equivalent to the actual effects of grazing seen in our 2014 study (10 g N/m2) and a much higher
level equal to that used by Cease et al. (2012) (17.5 g N/m2). Nitrogen was applied as an aqueous
solution of NH4NO3; with the control, unfertilised treatment receiving an equivalent volume of just
water. Plots were fertilized in early May and again at the end of June.

In late August 2015, we measured the density of E. unicolor within each treatment field plot. We
randomly placed one 1 m2 quadrat in each of the eighteen plots. Each quadrat was carefully
enclosed using a 1.5 m high shading screen barrier. All grasshoppers in each quadrat were then
removed using a sweep-net and the number of E. unicolor was counted (Heidorn & Joern, 1987). The
survey was conducted only on sunny days with minimal cloud cover and no wind, with three
replications. In early August 2015, we installed one cage into each of the treatment plots (18 cages in
total). We then introduced 14 fourth-instar nymphs (7 females and 7 males) to each cage, and
counted the survivors every three days for three weeks. We measured E. unicolor survival rate, body
size and mass gain in each cage using the methods described above.

In order to control for the potential confounding factors present in the field fertilisation
experiment, we simultaneously conducted a laboratory experiment to test the response of E.
unicolor to plant N content. In early August 2015, we collected fourth-instar nymphs from the field
and separated them according to sex. Then, we randomly assigned each nymph to one of three
treatments containing plant material that had received 0, 10 or 17.5 g N/m2. We stocked 10
individuals per sex per cage (in cages 0.08 m2 basal area × 0.3 m high, 36 cages in total, n =6). Fresh
L. chinensis grass was collected from each of the treatments from the field plots, offered in small
water-filled plastic vials, and replaced every other day. Grasshoppers were monitored for three
weeks, and then we measured grasshopper survival rate, body size and mass gain in each cage.

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 In mid-August 2015, we collected L. chinensis leaves from each of the field treatment plots, and
measured their N content, C content, toughness and water content using the same methods
Accepted Article
previously described. We also measured the volume of L. chinensis grass in 0.5 × 0.5 m quadrat
within each plot.

Data analyses

To assess the effects of cattle grazing on the response variables studied in the large herbivore
exclusion field experiment, we used linear mixed effects models, with ‘treatment’ included as a fixed
effect (2 levels: ‘grazed’ and ‘ungrazed’) and ‘replicate plots’ included as a random effect. The
specific response variables assessed were: performance variables (i.e. survival, body size and mass
gain) from male and female E. unicolor grasshoppers; plant biomass, cover, density, height, and
volume for each plant functional group separately and also across all groups combined (L. chinensis,
other grasses, and forbs); the microclimatic conditions (air temperature, air humidity and light
penetration) E. unicolor experienced in the plots; and the physical and chemical leaf traits of L.
chinensis grass (N content, C content, C:N ratio, toughness and water content). Models were fitted
using the function lmer from the package lme4 (Bates, Mächler, Bolker, & Walker, 2015) and the
package lmerTest was used to calculate P‐values (Kuznetsova, Brockhoff, & Christensen, 2016). To
examine the effect of cattle grazing on the response variable ‘abundance of E. unicolor grasshoppers’
over time, we added the fixed effect ‘sampling time’ (4 levels: the four sampling dates) the basic
linear mixed effect model outlined above.

For both N addition experiments in the field and in the laboratory, we used linear models to
determine the effects of the fixed factor ‘N addition treatment’ (3 levels: 0, 10 and 17.5 g N/m2) on
the response variables E. unicolor density and performance (i.e. survival, body size, and mass gain).
We also performed linear models with the fixed factor to assess the effects of ‘N addition treatment’
on L. chinensis leaf N content, C content, toughness, water content and volume. Tukey multiple
comparison analyses were used whenever significant differences were found.

All response variables were tested for homogeneity of variances, using the Levene's test. We
tested the normality of residuals using the Shapiro-Wilks test and the visual inspection of residuals
vs. fitted values (Zuur, Ieno, Walker, Saveliev, & Smith, 2009). The response variable ‘proportion of
surviving of E. unicolor in cages’ were arcsine square root transformed to ensure normality. We used
Satterthwaite's approximation to calculate the denominator degrees of freedom. Untransformed
data are presented in the figures. All statistical analyses were performed in the open source
software R 3.1.0 (R Development Core Team, 2014).

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 RESULTS
Accepted Article Effects of cattle grazing on E. unicolor population density and performance

Cattle grazing had a negative effect on the abundance of E. unicolor grasshoppers (grazing:
F1,28=47.54, P<0.001; Fig. 1), and the effect of grazing was consistent over time (grazing × time:
F3,28=0.96, P=0.43). E. unicolor abundance increased in both treatments throughout the growing
season before decreasing at the end of the season (time: F3,28=12.85, P<0.001).

Female E. unicolor survival rates were 44% lower in grazed than in ungrazed plots (F1,8=9.525,
P=0.015, Fig. 2a) and size (femur length) was reduced by 6% in grazed plots (F1,4=23.27, P=0.008; Fig.
2e), but mass gain was unaffected (Fig. 2c). Cattle grazing reduced mass gain of male E. unicolor (F1,8
=15.839, P=0.004; Fig. 2d), but did not affect their survival rate or femur length (Figs. 2b and 2f).

Effects of cattle grazing on vegetation and microclimatic conditions

Cattle grazing did not affect plant biomass or volume significantly for any of the studied plant
functional groups (Fig. 3, Appendix S2: Table S1). Cattle grazing did decrease the height of L.
chinensis by 6% (F1,8=6.034, P=0.04), but had no significant impacts on L. chinensis density or cover.
Grazing did not affect the height, density, or cover of the other grasses, the forbs, or the total of all
three plant groups (Appendix S2: Table S1).

Cattle grazing significantly increased air temperature by +0.77 ◦C on average at 35 cm above

ground (F1,4=14.266, P=0.019), but had no impacts on air temperature at 5 cm, air relative humidity
or PAR at any of the two levels above ground (Appendix S2: Table S2).

Cattle grazing significantly increased N content of L. chinensis leaves by 38% (F1,8=108.14,,

P<0.001; Fig. 4a), but did not alter leaf carbon content (Fig. 4b). This resulted in a 27% decrease in
C:N ratio of L. chinensis leaves (F1,8=75.992, P<0.001). In contrast, livestock grazing did not change
any of the plant physical traits assessed, with L. chinensis leaf toughness and water content
remaining similar in grazed and ungrazed plots (Appendix S2: Fig. S1).

Effects of nitrogen addition on L. chinensis grass and E. unicolor grasshopper

Fertilisation treatments in the field (10 g N/m2 and 17.5 g N/m2) significantly enhanced the N
content of L. chinensis leaves by 34 % and 122% compared to controls, respectively (F2, 15 =2852,
P<0.001; Appendix S2: Fig. S4a), but did not affect leaf carbon content (Appendix S2: Fig. S4b). These
changes led to 24% and 54% decreases in C:N ratios of L. chinensis leaves in 10 g N/m2 and 17.5 g

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 N/m2 treatments compared to the control treatment (F2, 15 =709.4, P<0.001; Appendix S2: Fig. S4c).
Fertilisation did not affect L. chinensis volume significantly, although a positive trend was observed
Accepted Article
(Appendix S2: Fig. S4d). In contrast, fertilisation significantly decreased L. chinensis leaf toughness
(F2,15 = 17.24, P<0,001). There was some evidence for an effect of N addition on water content (F2,15
=3.962, P=0.04), but Tukey multiple comparison analysis was not significant (see Appendix S2: Fig.
S4e, f).

Nitrogen addition of 10 g N/m2 and 17.5 g N/m2 markedly decreased E. unicolor density within
each field plot by 44% and 51%, respectively (F2,15 =4.18, P=0.036; Appendix S2: Fig. S2). In addition,
fertilisation inputs significantly reduced E. unicolor performance. Specifically, survival rate and femur
length of female E. unicolor were reduced in N-enriched plots (survival rate, F2,15 =11.61, P<0.001;
femur length, F2,15 =7.019, P=0.007; Figs. 5a and 5e), while mass gain was unaffected (Fig. 5c).
Similarly, femur length of male E. unicolor was also reduced in fertilised plots (F2,15 =6.331, P=0.01;
Fig. 5f), while enrichment did not affect the survival rate or mass gain of male E. unicolor (Figs. 5b
and 5d).

Consistent with results in the field fertilisation experiment, E. unicolor performance was also
reduced when fed N-fertilised plant material in laboratory conditions. Survival rate, mass gain and
femur length of female E. unicolor were significantly lower when fed N-fertilized L. chinensis material
(survival rate, F2,15 =13.69, P<0.001; mass gain, F2,15 = 4.352, P=0.032; femur length, F2,15 =7.803,
P=0.005; see Appendix S2: Fig. S3a, c, e). Again, the survival rate and femur length of male E. unicolor
were not affected by N enrichment, while mass gain of male E. unicolor was significantly reduced
when fed fertilised plant material (F2,15 =16.08, P<0.001; Appendix S2: Fig. S3d).

DISCUSSION

Inter-specific interactions between closely related herbivorous species have been widely studied and
typically result in negative effects for both species as a result of the exploitation of a limiting shared
plant resource (i.e. competition). As an example, competitive interactions have been found between
wild ungulates and cattle (Odadi et al., 2011), and grasshoppers and caterpillars (Xi, Griffin, & Sun,
2013). In contrast, the outcomes of interactions between distantly related herbivores with disparate
body sizes are complex and tend to be asymmetrical (Gómez & González-Megías, 2002). This study
demonstrates that large vertebrate grazing produces a significant negative effect on a much smaller
invertebrate herbivore in a grassland ecosystem. We simultaneously explored multiple potential
driving factors underlying this response, including plant quality and quantity, microclimate, and
vegetation structure. We found small but significant effects of vertebrate grazing on grass height and
canopy air temperature, but a comparatively large 38% increase in plant N content. We were able to

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 reproduce the reduction in grasshopper abundance and performance by using N addition to mimic
the effects of cattle grazing, which enhanced plant N-content and volume while reducing leaf
Accepted Article
toughness. We further isolated the contribution of plant N content from potentially confounding
shifts in plant species composition, plant structure, or microclimate conditions by feeding clipped
leaves to grasshoppers in the lab. Therefore, while we do not exclude the possibility of other
contributing factors, we conclude that a change in plant quality (N content) of the shared host grass
appears to be the major driver of the effect of cattle on grasshoppers.

Counter-intuitively, the negative effect of cattle on grasshoppers appeared to be the result of an

increase in plant N content in cattle grazed areas. Although high leaf N content is often thought to
be beneficial for herbivores (Mattson, 1980), our experiments show that N enrichment due to
moderate cattle grazing resulted in lower survival rates and reduced performance for the
herbivorous grasshopper. Cease et al. (2012) also found that N fertilisation and an increase in
protein: carbohydrate ratio negatively affected grasshopper performance.

Several potential mechanisms may explain the negative effect of elevated plant N content on
grasshopper performance. Firstly, organisms are predicted to have specific dietary intake targets for
each nutrient (i.e., the geometric framework, Raubenheimer & Simpson, 1993), and a mismatch
between an organism’s intake target and the nutrient content of its food may result in a surplus or a
deficit of particular elements (Behmer & Joern, 2008; Ibanez, Millery, D'Ottavio, Guilhot, & Vesin,
2017). In our study, cattle grazing (as well as fertilisation) significantly decreased leaf C:N ratio,
potentially producing an imbalance in nutrients available to E. unicolor grasshoppers. Secondly,
nutrient-rich plants may reduce insect performance by affecting specific physiological processes such
as increasing the metabolic costs of storing and excreting excess N (Boersma & Elser, 2006). There is
indirect evidence that such a mechanism does operate in aphids (Zehnder & Hunter, 2009). Finally,
some physical and chemical plant traits, such as N-based alkaloids, could change concomitantly with
N content (Vannette & Hunter, 2011). In our system, the increased N content in L. chinensis leaves
from grazed plots did not correlate with changes in leaf toughness or water content, while leaves
from N-addition plots had lower leaf toughness, suggesting that at least physical leaf traits were not
responsible for the negative response of E. unicolor grasshoppers. Grasses generally lack levels of
secondary metabolites necessary for effective chemical defence (Mattson, 1980; Tscharntke &
Greiler, 1995), but some studies have demonstrated that grasses infected by an endophytic fungi
(family Clavicipitaceae) could produce a variety of alkaloid compounds deterrent to herbivores (Clay,
2014). The study specific to endophytes in L. chinensis showed that the endophyte infection rate of
L. chinensis is low, and currently there is no evidence suggesting endophytes can facilitate Leymus
grass through production of alkaloids (Wang et al., 2016). Further detailed experiments are needed
to clarify the plausibility of this mechanism.

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 Over an evolutionary time scale, we suggest that the negative effects of nutrient rich resources
on E. unicolor population density and performance is most likely a result of their long-term
Accepted Article
adaptation to an N-limited environment. Many fertilisation experiments in the Eurasian steppe have
documented that the dominant grass L. chinensis is limited by N (Wang et al., 2017). Hence, E.
unicolor might have specialised to exploit this nutrient-poor resource. Given that many grassland
ecosystems are similarly low in N content, we surmise that the mechanisms observed in this study
might be widespread among insect herbivores feeding on low-N grasses. For example, many
grasshopper species such as Phoetaliotes nebrascensis in North America (Joern & Behmer, 1998),
Chortoicetes terminifera in Australia (Clissold et al., 2006), and Oedaleus asiaticus in north Asia
(Cease et al., 2012) have a preference for low quality grasses instead of other more nutrient-rich
forbs and legumes, which might be a consequence of their adaptation to N-limited environments.

Although we conclude that plant quality (N content) is a major driver of the effect of cattle on
grasshoppers, we cannot rule out the possibility that changes to microclimatic conditions and plant
structure also play a role (Suominen et al., 1999; van Klink et al., 2015). We documented a modest
increase in air temperature of 0.77 ºC at 35 cm above ground in grazed plots. Although a similar
magnitude increase in temperature of 1ºC had no observable effect on grasshopper performance or
survival in a previous study, negative effects of temperature on grasshopper survival and
performance were documented in response to substantial increases of 3ºC (Laws & Belovsky, 2010).
Cattle also reduced grass height by 6%, suggesting that a reduction in plant structure may have
contributed to the negative response of grasshoppers to grazing. However, grasshoppers also
responded negatively to N-addition, which tended to increase plant volume. Overall grass
abundance was unaffected by cattle, despite a decrease in plant height, suggesting compensatory
regrowth following moderate grazing intensity (McNaughton, 1979). Previous studies in a similar
system reported that L. chinensis is a highly grazing-tolerant species that can compensate or
over-compensate losses under moderate simulated grazing situation (Gao et al., 2008). Grazing may
also improve conditions for plant growth by reducing intraspecific competition for light and
increasing nutrient availability directly by adding readily accessible N in the form of urine and faeces
or indirectly by decreasing plant litter (Liu et al., 2017). Furthermore, grazed plants have been shown
to increase chlorophyll and photosynthetic enzymes concentration in remaining or new leaves,
which consequently results in a higher total N concentration (Briske, 1996; Price, 1991). Indeed,
plant nutrient enrichment is a common result of large herbivore grazing and has been reported in
many ecosystems including forests (Riipi et al., 2005) and African savannas (McNaughton, 1976) and
alpine meadows (Cao et al., 2015). However, it is noted that the effect of large vertebrates on grass
N content may vary based on the intensity of grazing. Interestingly, Cease et al. (2012) found a
positive effect of large vertebrate herbivores on grasshoppers in the Eurasian steppe system
because heavy livestock grazing resulted in lower grass N-content, likely due to erosion and the loss

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 of soil organic N. Besides grazing intensity and frequency, the effect of large vertebrate herbivory on
plant quality also depends on several intrinsic and extrinsic factors, such as plant resistance strategy,
Accepted Article
plant life history as well as environmental conditions (Briske, 1996).

Invertebrate herbivores, particularly specialist grasshoppers and caterpillars, usually reach

maximal performance within a fairly limited range of plant nutrients (Behmer, 2009). The optimal
range of each nutrient for a given herbivorous species can be highly variable, which is most notable
between different insect species (Behmer & Joern, 2008), but it also occurs within a species as a
result of developmental status, immediate physiological state and/or sex (Hawlena & Schmitz, 2010).
Our results strongly suggest that large vertebrate herbivores (cattle) exert a negative indirect effect
on small insect herbivores (E. unicolor grasshopper) whereby cattle modify plant quality, which
decreases grasshopper population densities, survival and performance. Therefore, understanding
multiple pathways whereby large herbivores drive invertebrate population merits further
exploration and can provide new insights into indirect interactions between distantly related and
differently sized herbivores.

CONCLUSIONS

Although large vertebrate herbivores are widely known to exert strong effects on invertebrate
herbivores in terrestrial ecosystems, many questions surrounding these biotic interactions remain to
be answered due to the paucity of carefully designed controlled experiments. Our study provides
direct evidence for a mechanistic understanding of a competitive interaction that might be pervasive
between differently-sized herbivores, even in ecosystems with very light grazing regimes. Uniquely,
we show that a change in plant quality appears to be an important driver of these competitive
interactions. These less obvious but relevant pathways warrant more attention, given their potential
generality. These findings further extend our understanding of the mechanisms for herbivore
coexistence in natural ecosystems, and may have important implications for developing effective
grazing management schemes.

ACKNOWLEDGEMENTS

We thank Y. Wang, H. Li and K. Y. Wang for help with the field work, O. J. Schmitz, T. R. Seastedt, M.
Huntzinger for suggestions on the early draft of this manuscript. We also thank two anonymous
reviewers for their helpful comments on the manuscript. This project was supported by the National
Key Research and Development Program of China (2016YFC0500602), the National Natural Science
Foundation of China (No. 31230012, 31302012, 31700357), the Program for Introducing Talents to
Universities (B16011), the Program for Innovative Research Team in University (IRT-16R11), and the
Fundamental Research Funds for the Central Universities (2412016KJ023). J. F. Pagès acknowledges

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 financial support from the Welsh Government and Higher Education Funding Council for Wales
through the Sȇr Cymru National Research Network for Low Carbon, Energy and Environment.
Accepted Article
AUTHORS CONTRIBUTIONS

Y.Z. and Z.Z. contributed equally to this work. Y.Z., Z.Z., D.W. designed experiments; Y.Z., Q.M.
performed the experiments; Y.Z., Z.Z., J.F.P., D.F. analyzed the data; Y.Z., Z.Z., J.F.P., D.F., D.W., N.H.,
H.Z., L.W. drafted the manuscript; all the authors contributed to the interpretation of results and to
revise the manuscript critically.

DATA ACCESSIBILITY

Data available from the Dryad Digital Repository: https://doi.org/10.5061/dryad.6n5g482 (Zhu et al.,
2018)

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 FIGURE LEGENDS
Accepted Article FIGURE 1 Population density of grasshop
pper Euchorthippus unicolor in the grazed and ungrazeed plots
over 4 sampling dates in August and Septtember 2014. Values are means ± SE.

male grasshopper Euchorthippus unicolor in the grazed and

FIGURE 2 Performances of female and m
ungrazed plots in August 2014: (a, b) survvival rate, (c, d) mass gain, (e, f) body size. Seven femaale and
seven male 4th stage nymphs were monittored in field circular cages installed on each plot for tthree
weeks. Values are means ± SE. The data were
w analysed separately by sex, because female
grasshoppers are significantly larger than
n males. Asterisks indicate a significant difference (* = P <
0.05, ** = P ≤ 0.01, ***P ≤ 0.001).

FIGURE 3 Biomass of (a) all plants combin

ned, (b) Leymus chinensis, (c) other grasses, and (d) fo
orbs in
the grazed and ungrazed plots in August 2014. The difference between treatments in none of the
four panels was significant. Values are m
means ± SE.

FIGURE 4 Total (a) nitrogen content (perccentage dry mass) and (b) carbon content (percentagee dry
mass) of Leymus chinensis leaves in the grazed
g and ungrazed plots in August 2014. Asterisk ind
dicate
significant difference (* = P < 0.05, **P ≤ 0.01, ***P ≤ 0.001). Values are means ± SE.

FIGURE 5 Effects of nitrogen application on grasshopper Euchorthippus unicolor performance in field

circular cages. The (a, b) survival rate, (c, d) mass gain, (e, f) body size of female and male
grasshopper of different nitrogen applicaation treatments (0, 10 and 17.5 g N/m2). In August 20
015,
seven female and seven male 4th stage nyymphs were monitored for three weeks. Different lettters
above the columns indicate significant diifferences (P < 0.05). Values are means ± SE.

FIGURE 1
 FIGURE 2
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FIGURE 3
 FIGURE 4
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FIGURE 5

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