Content uploaded by Ling Wang
Author content
All content in this area was uploaded by Ling Wang
Content may be subject to copyright.
Available via license: CC BY 4.0
Content may be subject to copyright.
Plants Can Benefit from Herbivory: Stimulatory Effects of
Sheep Saliva on Growth of
Leymus chinensis
Jushan Liu
1
, Ling Wang
1
*, Deli Wang
1
*, Stephen P. Bonser
2
, Fang Sun
1
, Yifa Zhou
1
, Ying Gao
1
, Xing
Teng
1
1Key Laboratory of Vegetation Ecology, Ministry of Education, Institute of Grassland Science, Northeast Normal University, Changchun, Peoples Republic of China,
2Evolution and Ecology Research Centre, School of Biological, Earth and Environmental Sciences, University of New South Wales, Sydney, Australia
Abstract
Background:
Plants and herbivores can evolve beneficial interactions. Growth factors found in animal saliva are probably
key factors underlying plant compensatory responses to herbivory. However, there is still a lack of knowledge about how
animal saliva interacts with herbivory intensities and how saliva can mobilize photosynthate reserves in damaged plants.
Methodology/Principal Findings:
The study examined compensatory responses to herbivory and sheep saliva addition for
the grass species Leymus chinensis in three experiments over three years. The first two experiments were conducted in a
factorial design with clipping (four levels in 2006 and five in 2007) and two saliva treatment levels. The third experiment
examined the mobilization and allocation of stored carbohydrates following clipping and saliva addition treatments. Animal
saliva significantly increased tiller number, number of buds, and biomass, however, there was no effect on height.
Furthermore, saliva effects were dependent on herbivory intensities, associated with meristem distribution within perennial
grass. Animal saliva was found to accelerate hydrolyzation of fructans and accumulation of glucose and fructose.
Conclusions/Significance:
The results demonstrated a link between saliva and the mobilization of carbohydrates following
herbivory, which is an important advance in our understanding of the evolution of plant responses to herbivory. Herbivory
intensity dependence of the effects of saliva stresses the significance of optimal grazing management.
Citation: Liu J, Wang L, Wang D, Bonser SP, Sun F, et al. (2012) Plants Can Benefit from Herbivory: Stimulatory Effects of Sheep Saliva on Growth of Leymus
chinensis. PLoS ONE 7(1): e29259. doi:10.1371/journal.pone.0029259
Editor: Gustavo Bonaventure, Max Planck Institute for Chemical Ecology, Germany
Received October 6, 2011; Accepted November 23, 2011; Published January 3, 2012
Copyright: ß2012 Liu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the State Key Basic Research Program (2007CB106801), State Agricultural Commonweal Project (200903060-2, 201003019),
and the National Natural Science Foundation of China (No. 7 30571318, 30600427, 30590382). The funders had no role in study design, data collection and
analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: wangd@nenu.edu.cn (DLW); wangl890@nenu.edu.cn (LW)
Introduction
Herbivory can limit the growth and survivorship of plants, and
plants have evolved complex responses to avoid being consumed
and/or to survive and flourish after herbivory. It is widely
accepted that plants can tolerate physical and biotic stresses and
damage [1,2]. Plant compensatory growth is ubiquitous in nature
and an important adaptive response to herbivory [3,4]. There is
some experimental evidence that herbivory may stimulate plant
growth and increase plant fitness [5,6,7]. However, compensation
(and overcompensation) responses are not consistent across species
or environments. It has been demonstrated that plant response to
herbivory is species specific and compensation to herbivory is
specific to herbivory type and intensities [8,9,10,11,12]. So studies
are required to establish the environmental cues plants use to
initiate a compensation response. Animal saliva may be an
important cue plants use to stimulate growth and initiate
compensation [13,14,15].
Vittoria and Rendina (1960) originally suggested that grazers
caused plant growth stimulation by depositing saliva during
grazing, and later tests supported this hypothesis [16,17,18].
However, there are some studies demonstrating that herbivore
saliva had no, or even negative impacts on plants [19,20,21,22]
The positive impacts appear possible in view of growth regulators
in salivary systems of insects, such as cytokinins, auxins, and
jasmonic acid [9,12,23,24], and various growth factors in
mammalian submaxillary glands, including thiamine, nerve
growth factor (NGF), transforming growth factor (TGF) and
epidermal growth factor (EGF) [25]. Growth factors can intervene
directly in cellular metabolism by promoting differential tran-
scription of genes, so they may be expected to have activity in a
variety of organisms [26]. Jasmonate was found to be involved in
tuber size regulation by mediating cell expansion, which was
correlated with increased accumulation of sucrose [11,12].
Thiamine is a plant growth factor produced in shoots that is
necessary for root growth [27]. Dyer and Bokhari (1976) reported
grasshoppers might inject growth-promoting substance into
Bouteloua gracilis and stimulated tiller production. Mouse and
human EGF were found to enhance plant growth rate and
promote cell division of epicotyl [28,29].
Recent research in woody plants demonstrates that animal
saliva tended to stimulate branching [30,31]. The activation of
dormant meristems is crucial for compensatory growth following
herbivory, especially for branching in woody plants or tillering in
PLoS ONE | www.plosone.org 1 January 2012 | Volume 7 | Issue 1 | e29259
grasses [32]. Responses in these growth forms both arise from
outgrowth of axillary meristems after releasing of apical domi-
nance, which is under genetic and hormonal control [33]. On the
grassland of Inner Mongolia, Zhang et al. (2007) studied the effects
of sheep saliva on a semi-shrub and herbaceous species, and found
that sheep saliva stimulated tillering of herbaceous grass [34].
Plant response varies with herbivory intensities, where plants tend
to perform better under light herbivory intensity [8,14,35]. At light
herbivory intensity, there is large possibility for plants to
overcompensate for tissue loss, and animal saliva may be one of
the mechanisms behind overcompensation [13,14]. Plant regrowth
after herbivory depends on the availability and remobilization of
carbon reserve, and the availability of reserve meristems to be
allocated to new growth [36,37]. The different availability of
carbon and meristem reserve is responsible for the nonlinear
response of plant to herbivory intensities [2,32,38,39]. Despite of
some research on plant response to animal saliva, the mechanism
behind the response remains uncertain. No study has examined
the role of animal saliva in inducing plant compensatory growth
after herbivory damage, how the effects of saliva vary with
herbivory intensities and how saliva affects resource allocation
during regrowth after herbivory.
We conducted experiments to test the role of sheep saliva in
promoting compensatory responses to herbivory and mobilization
of stored resources in the perennial grass Leymus chinensis.We
hypothesized that: (1) saliva has largest effects at light herbivory
intensities, and (2) animal saliva could promote mobilization of
stored carbon reserve.
Methods
Ethics Statement
No specific permits were required for this study, because the
performance of this study was in accordance with guidelines set by
the Northeast Normal University. No specific permits or approval
was required for the animal work, because the care of sheep in the
studies was in accordance with relevant national and international
guidelines. To collect saliva, we put a cake of sponge into sheep
mouth when they chewed grasses. After about two minutes, the
sponge was taken out. All the performance was softly conducted by
hand, without any hurt or damage on the animals. No specific
permits were required for the described field studies, because the
field is owned by Northeast Normal University and the Songnen
Grassland Ecological Research Station performs the management.
No specific permits were required for these locations/activities,
because the location is not privately-owned or protected in any
way and the field studies did not involve endangered or protected
species.
Species and sites
We conducted three experiments at the Songnen Grassland
Ecological Research Station of Northeast Normal University, Jilin
Province, PR China (44u459N, 123u459E). There is a semi-arid
and continental climate with a frost-free period of about 140 days,
with annual mean temperature ranging from 4.6uC to 6.4uC and
annual precipitation from 290 to 450 mm. The main vegetation
type is meadow steppe predominated by Leymus chinensis and Stipa
baicalensis [40].
L. chinensis is a perennial rhizomatous grass with good
palatability and high forage value [8,40,41,42,43,44]. It is widely
distributed in the eastern region of the Eurasian steppe zone as a
dominant species from arid to semi-arid steppes in northern China
and eastern Mongolia, and it has extensive plasticity in
morphological and physiological characteristics. L. chinensis is a
clonal perennial grass with large belowground bud bank.This
species has the capacity of rapid regrowth after grazing or mowing
early in the season, and high tolerance to drought, cold and alkali
stresses [45,46]. Highly branched rhizomes lie horizontally about
5–15 cm beneath the soil surface, and the long rhizomes can
spread and form near monocultural stands.
Culture of experimental plants
At the beginning of May 2006, 2007 and 2008, seeds of L.
chinensis collected from the study area were germinated in bunched
paper cylinders (2 cm in diameter, 5 cm deep) which were filled
with soil to about 4 cm in depth and covered with 1 cm of soil
again after seeds were sprinkled in cylinders. Cylinders were kept
in a greenhouse and watered daily. At about 30 days of age, 13
seedlings of similar size plants per pot, were transplanted into
outdoor plastic pots (20 cm in diameter and 15.5 cm deep) filled
with 14 cm field soil in 2006, and in 2007 with the mixture of field
soil and fertile soil from commercial source in a 6:1 ratio. In the
two experiments, grasses were watered daily. In 2008, seedlings
were transplanted into pots filled with sand and watered with 1/7
strength Hoagland’s solution every day.
Saliva collection and application
Saliva was collected by inserting a cake of sponge into the
mouth of a sheep. The sheep chewed on the sponge for two
minutes and the sheep saliva was squeezed into a tube. After being
filtered by sponge, saliva was clean and there was no plant material
mixed in. For the saliva addition treatment in each of the
experiments, we clipped grasses and immediately applied saliva
with a mini brush across the cut end of the leaves (clipped plants)
or along the length of the leaf blades (non-clipped plants). The
sponge, tube and brush were sterilized with 75% alcohol and dried
before used.
Experimental design and measurements
Experiment 1, effects on plant growth. We performed the
first experiment from 18 July to 18 September, 2006. About one
month after being transplanted and adjusted to the outdoor
growing conditions, seedlings were assigned to one of four clipping
treatments (0, 25, 75 and 100% of above ground shoot height) and
one of two levels of saliva (with and without saliva at every clipping
level). In another experiment, we studied plant response to
different component of animal saliva, which showed that there was
no difference between clipping with- and without water
(unpublished data). Therefore, in present study we focused on
the difference between clipping with- versus without saliva, and
there was no clipping with water as control. There were 5 replicate
pots per treatment, and plants were harvested one month after
treatments. This resulted in a total of 40 pots in the experiment (4
clipping62 saliva65 replicates). Plants were randomly assigned to
all treatments and 5 ml sheep saliva per seedling was added to
saliva treated plants. All treatments were performed within about
2 hours, alternating between the two kinds of treatments at every
clipping level (clipping alone versus clipping with saliva) so as to
prevent any temporal bias. For every treatment, 10 shoots per pot
were randomly marked with wire rings, to measure shoot height.
For nondestructive sampling, we measured the height of the
marked shoots and counted the amounts of tillers in all the pots for
every treatment on 17 August. After measuring height and tiller
number, we harvested the grasses and counted the number of
buds. Grasses were separated into above- and belowground parts,
and oven dried at 70uC for more than 72 hours prior to measuring
biomass. The belowground tissue was carefully washed prior to
drying.
Plant Response to Animal Saliva
PLoS ONE | www.plosone.org 2 January 2012 | Volume 7 | Issue 1 | e29259
Experiment 2, effects on plant growth. The second
experiment was conducted from 13 July to 20 August 2007. The
design was similar to the first one except that there were 5 clipping
levels (0, 25, 50, 75 and 100%) and 6 replicates for every treatment
combination. The measurements and samplings were also
performed one month after treatments (20 August), and there
were 60 pots in total.
Experiment 3, response in carbohydrate mobili-
zation. Third experiment was conducted from 4 to 14 August,
2008. Thirty six pots of grasses were randomly allocated to two
treatments, clipping without saliva, and clipping with saliva. There
were three replicates for each treatment. All the plants were
clipped at 25% of shoot height. In clipping with saliva treatment,
plants were applied with sheep saliva immediately after clipping.
After 0, 0.5, 1, 3, 5 and 10 days of regrowth, three pots of plants in
every treatment combination were harvested and divided into
leaves, stems, rhizomes and fibrous roots, frozen in liquid nitrogen,
stored at 280uC and used for analysis of water-soluble
carbohydrate.
One hundred milligrams of frozen-dried plant tissue was
sampled from harvest plants and ground. A fine powder was
boiled in 4 ml 80% ethanol and extracted for 1 hour at 80uC. The
sample was centrifuged at 10,000 gfor 10 min after ethanol
extraction, and then the supernatant was preserved. Ten millilitre
of water was added to the pellet and the tube contents were mixed
and incubated for 1 hour at 90uC. After the aqueous extraction,
the sample was centrifuged at 10,000 gfor 10 min. Then the
supernatant was preserved and the aqueous extraction was
repeated once again with the pellet. The three supernatants were
pooled and evaporated to dryness. The residue was dissolved in
2 ml water, pooled and filtered with a 0.45-mm nylon membrane.
Aliquots of carbohydrate extract were passed through a column
containing cation-exchange resin (Dowex 50W X8-400 H
+
-form;
Sigma) and a column filled with anion-exchange resin (Amberlite
CG-400 II; Fluka) to remove charged compounds. Purified
carbohydrates were separated and quantified by high-performance
liquid chromatography (HPLC) on a Sugar-PAK column
(300 mm long, 6.5 mm i.d.; Millipore Waters), eluted at
0.5 ml min
21
and 85uC with 0.1 mM CaEDTA in water, using
mannitol as internal standard and a refractometer as a sugar
detector [47].
Statistical analysis
For plant growth variables in Experiment 1 and 2, we
performed two-way factorial ANOVA to evaluate the effects of
clipping and saliva at every sampling time, with saliva, clipping
and their interaction as fixed factors. Tukey-Kramer test was
followed to examine the difference among clipping levels.
Moreover, Bonferroni correctiont-test was carried out to compare
the difference between treatments with- versus without saliva at
every clipping level, in whici the ‘‘p’’ value for each test was equal
to alpha divided by the number of test (n =4 in Experiment 1 and
n = 5 in Experiment 2). Variables were log transformed, where
necessary, to meet the assumptions of statistical analyses. In
Experiment 3 to assess the effects of treatments on carbohydrate
and how they varied with time, a repeated measures analysis of
variance (ANOVA) was also used with clipping without- versus
clipping with saliva as between-subject factor (main effect), and
time as within-subject (repeated) factor. Bonferroni correction was
carried out to analyze the difference between treatments (clipping
without- versus with saliva) at every time, and the ‘‘p’’ value was
adjusted based on the number of test (n = 6). All statistical analyses
were conducted in SAS 9.0 (SAS Institute, Cary, NC, USA).
Results
Growth responses to clipping and saliva
Shoot elongation. Both in 2006 and 2007, plant height
decreased with increasing clipping intensities (Fig. 1, Table 1). In
the two experiments, there was neither significant saliva effect nor
interactive effect between saliva and clipping (Table 2), although
there was a trend towards an increase when saliva was applied to
clipped shoots (Fig. 1).
Accumulation in biomass. In 2006, above- and
belowground biomass decreased with increasing clipping
intensities, except that plant compensated in above ground
biomass at 25% clipping level (Table 1). In 2007, there was no
difference among 0%, 25% and 50% clipping treatments, and at
75% and 100% clipping treatments biomass decreased
significantly (Table 1). At 25% clipping level, adding saliva on
clipped shoots significantly increased aboveground biomass on
2006 and 2007 (Fig. 1). At 100% clipping level, in contrast to
clipped plants without saliva, the clipped and saliva-applied grasses
produced significantly more above- and belowground biomass in
the two years (Fig. 1), and saliva had significant effects on above
and belowground biomass (Table 2)
Dynamics of tillering. In experiment 1, the 100% clipping
treated plants had significantly fewer tillers than the other clipping
treatments, whereas, in experiment 2, there was no difference
among clipping treatments (Table 1, 2). In 2006, at 100% clipping
level, the clipped and saliva applied plants had significantly more
tillers than the grasses clipped without saliva, whereas, in 2007, the
overall saliva effect resulted from the significant increase in tillers at
25% and 100% clipping level (Fig. 1). No significantly interactive
effect between saliva and clipping was found (Table 2).
Changes in the bud bank. In 2006, plants at 0% and 25%
clipping levels had significantly more buds than those at 75% and
100% ones (Table 1). In 2007, clipping effects came only from the
difference between 100% clipped grasses and those at other clipping
levels (Table 1). All the saliva effects and interactive effects with clipping
resulted from the difference between clipping with and without saliva at
25% (2006), or at 25% and 100% clipping levels (2007) (Table 1, Fig. 1).
Carbohydrate mobilization
Hydrolyzation of fructans and changes in suc-
rose. During the first 3 days of experiment 3, fructans in all
tissues fell rapidly and remained constant at low level thereafter,
except that in the first 0.5 days there was a slight increase in
aboveground tissues (Fig. 2a
1
to 2a
4
). For fructan contents, both
clipping and saliva effects (except in leaf) and interactive effects with
time (except in stem) were significant. Furthermore in every
component tissue, clipped and saliva treated plants had
signficantly lower content of fructans, compared to clipped grasses
without saliva (Table 3, Fig. 2a
1
to 2a
4
). This demonstrates that
saliva promoted fructans hydrolization in plant tissues. In
aboveground tissues, sucrose concentration increased rapidly
during the first day of regrowth following treatments, and then it
declined rapidly in the following two days. Thereafter, sucrose
content did not change significantly in leaf and stem (Fig. 2b
1
and
2b
2
), whereas in belowground parts, sucrose content increased
gradually until the end of the experiment, except that in rhizome it
declined 10 days after treatments (Fig. 2b
3
). There was no significant
difference between treatments in sucrose (Table 3).
Accumulation of glucose and fructose. In the first day of
the experiment, glucose content did not change significantly
(Fig. 2c
1
to 2c
4
), except in leaf tissue where glucose decreased by
about 50%. During the following period glucose contents
increased gradually until the end of the experiment, except that
Plant Response to Animal Saliva
PLoS ONE | www.plosone.org 3 January 2012 | Volume 7 | Issue 1 | e29259
in rhizome it did not significantly change after day 3. In
aboveground tissues, fructose contents decreased at the onset of
experiment, and increased gradually thereafter (Fig. 2d
1
and 2d
2
).
In rhizome tissue, there was a similar change in fructose content
except that fructose began to accumulate after a lag time of 2 days
(Fig. 2d
3
). In fibrous root tissue, fructose content of clipped plants
with saliva did not vary at the beginning of experiment, increased
on day 1 and further after 5 and 10 days (Fig. 2d
4
), whereas in
clipped without saliva plants, there was only an increase 3 days
after treatments. For monosaccharides, in aboveground tissues
difference between treatments was significant, and in belowground
parts both treatment effects (except in rhizome) and interactive
effects with time (except fructose in rhizome) are significant
(Table 3). Clipped and saliva applied plants had slightly more
glucose and fructose than clipped plants without saliva (Fig. 2c
1
to
2d
4
), which suggested that saliva stimulated monosaccharides to
accumulate in grasses.
Discussion
Saliva effects on tillering
Results from the first two experiments showed that sheep saliva
increased the number of tillers, and the number of buds, which
Figure 1. Effects on regrowth. The effects of clipping and saliva on height, aboveground biomass, belowground biomass (BGB), tillers and buds
(back-transformed from the log scale) of Leymus chinensis both on 17 August 2006 and 20 August 2007. There are four clippinglevels (0%, 25%, 75% and
100% of aboveground shoots) in 2006 and five ones (0%, 25%, 50%, 75% and 100%) in 2007. Bars represent standard errors. **, P,0.05; *, P,0.01.
doi:10.1371/journal.pone.0029259.g001
Table 1. Results of Duncan multiple comparisons of
differences in height, aboveground biomass (AGB),
belowground biomass (BGB), tillers and buds (back-
transformed from the log scale) among clipping levels both in
2006 and 2007.
Height AGB BGB Tillers Buds
2006 0% 2.87a 1.00a 1.58a 3.53a 3.80a
25% 2.74ab 1.07a 1.60a 3.63a 3.91a
75% 2.67b 0.89a 1.37a 3.54a 3.59a
100% 2.32c 0.39b 0.67b 3.27b 2.72b
2007 0% 3.40a 1.82a 1.60ab 4.11a 4.29a
25% 3.36a 1.81a 1.65ab 4.14a 4.35a
50% 3.35a 1.79a 1.71a 4.21a 4.19a
75% 3.23b 1.61b 1.51b 4.17a 4.26a
100% 2.98c 1.27c 1.17c 4.19a 3.45b
Different letters indicate statistical significance at P,0.05 (n = 5 in 2006, n = 6 in
2007).
doi:10.1371/journal.pone.0029259.t001
Plant Response to Animal Saliva
PLoS ONE | www.plosone.org 4 January 2012 | Volume 7 | Issue 1 | e29259
Table 2. Results of two ways ANOVA for the effects of saliva, clipping and their interaction on height, aboveground biomass
(AGB), belowground biomass (BGB), tillers and buds both in 2006 and 2007.
Height AGB BGB Tillers Buds
Time Treatments df
FP FP FP FP FP
2006 Saliva 1 3.80 0.0691 1.81 0.1897 2.50 0.1269 4.73 0.0377
*
0.29 0.595
Clipping 3 13.74 0.0001
**
64.54 0.0001
**
41.67 0.0001
**
11.30 0.0001
**
13.83 0.0001
**
Saliva6Clipping 3 0.37 0.7740 1.37 0.2736 2.07 0.1309 2.20 0.1090 3.91 0.0177
*
2007 Saliva 1 1.70 0.1989 7.96 0.0071
**
6.09 0.0179
*
7.94 0.0058
**
5.62 0.0224
*
Clipping 4 32.45 0.0001
**
14.95 0.0001
**
16.27 0.0001
**
0.97 0.4276 14.54 0.0001
**
Saliva6Clipping 4 0.36 0.8367 0.15 0.9618 1.88 0.1317 1.55 0.1943 2.78 0.0387
*
**, P,0.05;
*, P,0.01.
doi:10.1371/journal.pone.0029259.t002
Figure 2. Response of carbohydrate concentrations. The differences between clipping without (real line) and with saliva (broken line) in
carbohydrate concentrations, fructans (a
1
–a
4
), sucrose (b
1
–b
4
), glucose (c
1
–c
4
) and fructose (d
1
–d
4
) in component parts, leaf (a
1
–d
1
), stem (a
2
–d
2
),
rhizome (a
3
–d
3
) and fibrous root (a
4
–d
4
), within 10 days after treatments in 2008. Bars represent standard errors. **, P,0.05; *, P,0.01.
doi:10.1371/journal.pone.0029259.g002
Plant Response to Animal Saliva
PLoS ONE | www.plosone.org 5 January 2012 | Volume 7 | Issue 1 | e29259
could be considered as tillering potential (Fig. 1). The stimulatory
effect on tillering is similar to that on branching in woody plants
[30,31], which are both from reserve meristems after the removal
of apical dominance due to grazing or clipping [48]. For grasses,
vegetative buds and active meristems are of pivotal importance,
and successive tiller production by the development of axillary
buds allows persistence of perennial grasses [49,50]. Moreover,
increased branching or tillering is one of the main mechanisms of
compensatory growth and has been considered as one of
mutualistic relationships between grasses and grazers [32,51,52].
Before defoliation shoot apex suppresses lateral meristem growth,
in which auxins and cytokinins are involved and have opposite
effects, that is, auxins inhibit and cytokinins promote branch
growth [33,53]. Herbivory breaks apical dominance and activates
reserve meristem to outgrowth, increasing tillers [50]. Since it is
known that there is plant growth factors in animal saliva, saliva left
on plant during grazing could have a positive effect on plant
branching or tillering. Dyer and Bokhari (1976) found that plants
experiencing herbivory by grasshoppers were able to produce
more tillers than those that were simply clipped, and they
suspected that plants were affected by unidentified growth
regulators contained in herbivore saliva. Furthermore, they
suggested that growth-promoting substance was injected into
plant endogenous metabolic process and then translocated to
zones of tiller primordium [16]. Therefore, effects of animal saliva
on plant growth related to correlation between growth regulators
in saliva and meristematic tissue within plant, and are most
effective on branching in woody plants or tillering in grass, which
was confirmed in Zhang et al. (2007) and our results.
In 2006 and 2007, the application of sheep saliva had significantly
positive impacts on plant biomass (Fig. 1). This stimulation should
be attributed to the increased tillers. Tiller number increased
throughout the experiment but most new leaves on these tillers
remained unexpanded, and saliva had no effects on height in the
two experiments (Table 2). Thus, experimentally induced compen-
sation in biomass was due to an increased number of tillers.
Saliva effects and clipping intensity
In the first two experiments, saliva effects varied with clipping
levels. Specifically, saliva effects were greatest in the 25% and
100% clipping treatments. This effect was especially evident for
buds where saliva and clipping had significant interactive effects
(Table 2). We believe that these experimental effects are closely
associated with the location of meristems within a plant. Herbivory
tolerance and compensation often include regrowth by production
of new shoots through activation of dormant buds [32]. According
to meristem allocation models, the patterns of compensatory
regrowth responses following grazing depend on the number of
latent meristems that escape from being damaged, and the
activation sensitivity of meristems related to the degree of damage
[38,39]. The increased tillers are the result of outgrowth of buds at
the base of shoots and along the rhizomes (i.e. the location of the
active meristems). The dynamics of tillering is a product of the
availability and activity of basal meristems and the hormonal
activity of the apical meristems [33].
Animal saliva contains various growth factors [54] and several
plant growth regulators, such as cytokinins and auxins, have been
found in the salivary systems of insects [23,24]. These chemicals
may be transferred in feeding processes to influence both plants
and herbivore [20,28]. So, saliva addition should be most effective
when it is applied near the regions of active cell growth (i.e.
meristems) [16,55], and the magnitude of saliva effects on plant
growth should vary with location of herbivory damage. The point
of damage in the 25% clipping treatment is up close to the base of
apical meristems and young leaves, which exert apical dominance
[33], and undoubtedly, and the point of damage in the100%
clipping treatment is adjacent to basal meristems. The results
demonstrated that it was most effective for saliva to stimulate
tillering when applied at the two clipping height levels being closest
to either active or basal meristems, and it was shown that, in our
results, saliva had the highest positive effects when plants were
completely clipped (100%) (Fig. 1). As we hypothesized, the
stronger saliva impacts at light clipping intensity validated the
Table 3. Repeated measures ANOVA for between-subject effects, treatments (clipping without and with saliva), and within-subject
effects (repeated effects), time, and their interaction effects, time6treatments for carbohydrate (fructans, sucrose, glucose and
fructose) concentrations of component parts (leaf, stem, rhizome root and fibrous root) of Leymus chinensis.
Fructans Sucrose Glucose Fructose
df
FP FP FP FP
Leaf Time 5 110.76 0.0001
**
14.07 0.0001
**
88.60 0.0001
**
23.90 0.0001
**
Treatments 1 2.11 0.1589 1.96 0.1724 6.01 0.0203
*
5.21 0.0300
*
Time6Treatments 5 6.73 0.0004
**
6.58 0.0003
**
0.00 1.0000 0.00 1.0000
Stem Time 5 28.77 0.0001
**
33.01 0.0001
**
270.88 0.0001
**
82.54 0.0001
**
Treatments 1 6.18 0.0191
*
3.33 0.0789 14.52 0.0007
**
19.05 0.0002
**
Time6Treatments 5 2.15 0.0882 3.25 0.0201
*
0.18 0.9689 0.51 0.7689
Rhizome Time 5 32.87 0.0001
**
15.52 0.0001
**
168.76 0.0001 24.02 0.0001
**
Treatments 1 37.60 0.0001
**
0.47 0.4992 3.00 0.0954 4.04 0.0550
Time6Treatments 5 7.48 0.0001
**
0.97 0.4576 4.42 0.0051
**
1.83 0.1428
Fibrous roots Time 5 36.16 0.0001
**
39.05 0.0001
**
153.38 0.0001
**
74.88 0.0001
**
Treatments 1 31.89 0.0001
**
3.24 0.0822 23.61 0.0001
**
55.66 0.0001
**
Time6Treatments 5 6.20 0.0004
**
0.84 0.5323 4.78 0.0031
**
13.47 0.0001
**
**, P,0.05;
*, P,0.01.
doi:10.1371/journal.pone.0029259.t003
Plant Response to Animal Saliva
PLoS ONE | www.plosone.org 6 January 2012 | Volume 7 | Issue 1 | e29259
expectation that animal saliva played important role in plant
compensatory response at light herbivory intensities [13,14].
Saliva accelerates carbohydrate mobilization during
regrowth
In the third experiment 2008, our results indicated that clipping
stimulated the mobilization of fructans. For each part of plant, the
significant treatment (or time6treatment) demonstrated that (at
least for some of the time) the saliva treated plants were more
quickly mobilizing stored fructans. Similarly, glucose and fructose
were increasing (Table 3, Fig. 2). This suggested that photosyn-
thesis in the remaining tissues had increased, and the newly
produced tissues were photosynthesizing quickly to compensate for
the losses to herbivores. Once again the highly significant
treatment effects demonstrated that saliva treated plants had a
greater compensation response than untreated plants (Table 3,
Fig. 2).
Defoliation by grazing or clipping reduces the amounts of the
leaf surface and thereby supply and allocation of photosynthate
[56]. Consequently carbon supply to aboveground regrowth
depends transiently on carbon reserves in the whole seedlings. A
plant’s ability to rapidly regrow following damage is fundamental
to tolerance strategy to herbivory [36,57]. Soluble carbohydrate
reserves are often considered as primary source of carbon for
regrowth following defoliation, and rapid mobilization of reserves
is crucial. Results in the third trial exhibited that in every
component part, fructans were hydrolyzed, and glucose and
fructose accumulated after treatments (Fig. 2). This suggests that
the whole seedling was a source for resources and supplied carbon
for growth of new tissue and production of new tillers. The
manner in which resource allocation patterns shift in response to
damage is under hormonal control, and auxins may affect bud
outgrowth indirectly by mobilizing resource to already differen-
tiated meristems [58,59]. The impact of saliva on resource
mobilization is ascribed to the regulation of various growth factors
contained in saliva, which regulate plant growth and metabolism,
interacting with regulation by endogenous hormones in plants
such as jasmonic acid. Jasmonic acid is one of the products of
octadecanoid pathway, which are up-regulated in response to
herbivory damage [9]. Interestingly, jasmonates have been shown
to have multiple physiological functions, mediating cell expansion
and accumulation of sucrose in tuber [11].
Conclusions
Animal saliva effects on plant growth are much more complex
than previously thought. In this study, we found that animal saliva
stimulated growth of perennial grasses, accelerating mobilization
of photosynthate reserves, enhancing buds tillers and consequently
increasing biomass. There were evident physiological responses to
saliva application soon after treatments, however, saliva effects on
growth properties only occurred one month following treatments.
In the present study, we also show that saliva effects varied with
clipping levels, stronger at light and complete clipping level. This is
associated with meristem distribution within perennial grass,
which is adapted to grazing in the long term. The stimulatory
effects at light herbivory intensity favour plants to compensate or
overcompensate, consistent with the grazing optimization hypoth-
esis. Under intense grazing pressure, saliva contributes to
minimize herbivory damage. Saliva effects are beneficial for
plants to tolerate continuous herbivory and be adapted to grazing
in the long term, which provides insight into the interpretation of
mutualism and coevolution between plants and herbivores in
grazing systems.
Acknowledgments
We thank Y. Li, Y. Huang, S. Li, L. Li, L. Zhang and J. Wang for their
assistance in the field and lab.
Author Contributions
Conceived and designed the experiments: JL DW LW. Performed the
experiments: JL YG XT FS. Analyzed the data: JL YG. Contributed
reagents/materials/analysis tools: JL FS YZ. Wrote the paper: JL LW DW
YG SB.
References
1. Fornoni J (2011) Ecological and evolutionary implications of plant tolerance to
herbivory. Functional Ecology 25: 399–407.
2. Stowe KA, Marquis RJ, Hochwender CG, Simms EL (2000) The evolutionary
ecology of tolerance to consumer damage. Annual Review Of Ecology and
Systematics 31: 565–595.
3. Crawley MJ (1987) Benevolent herbivores? Trends in Ecology and Evolution 2:
167–168.
4. Lennartsson T, Tuomi J, Nilsson P (1997) Evidence for an evoluti onary history
of overcompensation in the grassland biennial Gentianella campestris (Gentiana-
ceae). American Naturalist 149: 1147–1155.
5. McNaughton SJ (1976) Serengeti migratory wildebeest: Facilitation of energy
flow by grazing. Science 191: 92–94.
6. Krebs CJ (2001) Ecology: The experimental analysis of distribution and
abundance Benjamin Cummings, an imprint of Addison Wesley Longman, Inc.
San FranciscoCalifornia, .
7. Owen DF, Wiegert RG (1976) Do consumers maximize plant fitness? Oikos 27:
488–492.
8. Gao Y, Wang DL, Ba L, Bai YG, Liu B (2007) Interactions between herbivory
and resource availability on grazing tolerance of Leymus chinensis. Environmental
and Experimental Botany.
9. Garrido E, Andraca-Go´mez G, Fornoni J (2011) Local adaptation: Simulta-
neously considering herbivores and their host plants. New Phytologist.
10. Gavloski JE, Lamb RJ (2000) Compensation for herbivory in cruciferous plants:
Specific responses to three defoliating insects. Environmental Entomology 29:
1258–1267.
11. Gavloski JE, Lamb RJ (2000) Specific impacts of herbivores comparing diverse
insect species on young plants. Environmental Entomology 29: 1–7.
12. Gavloski JE, Lamb RJ (2000) Compensation by cruciferous plants is specific to
the type of simulated herbivory. Environmental Entomology 29: 1273–1282.
13. Paige KN, Whitham TG (1987) Overcompensation in response to mammalian
herbivory: The advantage of being eaten. American Naturalist 129: 407–416.
14. McNaughton S (1979) Grazing as an optimization process: grass-ungulate
relationships in the Serengeti. American Naturalist. pp 691–703.
15. Poveda K, Go´mez Jime´nez MI, Kessler A (2010) The enemy as ally: Herbivore-
induced increase in crop yield. Ecological Applications 20: 1787–1793.
16. Dyer MI, Bokhari UG (1976) Plant-animal interactions: Studies of the effects of
grasshopper grazing on blue grama grass. Ecology 57: 762–772.
17. Matches AG (1992) Plant response to grazing: A review. Journal of production
agriculture 5: 1–7.
18. Vittoria A, Rendina N (1960) Fattori condizionanti la funzionalita tiaminica in
piante superiori e cenni sugli effetti dell bocca dei runinanti sull erbe pascolative.
Acta Medica Vet (Naples) 6: 379–405.
19. Johnston A, Bailey CB (1972) Influence of bovine saliva on grass regrowth in the
greenhouse. Canadian Journal of Animal Science 52: 573–574.
20. Reardon PO, Leinweber CL, Merrill LB (1974) Response of sid eoats grama to
animal saliva and thiamine. Journal of Range Management 27: 400–401.
21. Capinera JL, Roltsch WJ (1980) Response of wheat seedlings to actual and
simulated migratory grasshopper defoliation. Journal of Economic Entomology
73: 258–261.
22. Detling JK, Ross CW, Walmsley MH, Hilbert DW, Bonilla CA, et al. (1981)
Examination of North American bison saliva for potential plant growth
regulators. Journal of Chemical Ecology 7: 239–246.
23. Miles PW, Lloyd J (1967) Synthesis of a plant hormone by the salivary apparatus
of plant-sucking hemiptera. Nature 213: 801–802.
24. Engelbrecht L, Orban U, Heese W (1969) Leaf-miner caterpillars and cytokinins
in the ‘‘green islands’’ of autumn leaves. Nature 223: 319–321.
25. Cohen S (1962) Isolation of a mouse submaxillary gland protein accelerating
incisor eruption and eyelid opening in the new-born animal. Journal of
Biological Chemistry 237: 1555–1562.
26. McNaughton SJ (1985) Interactive regulation of grass yield and chemical
properties by defoliation, a salivary chemical, and inorganic nutrition. Oecologia
(Berlin) 65: 478–486.
Plant Response to Animal Saliva
PLoS ONE | www.plosone.org 7 January 2012 | Volume 7 | Issue 1 | e29259
27. Bonner J (1937) Vitamin B
1
a growth factor for higher plants. Science 85:
183–184.
28. Dyer MI (1980) Mammalian epidermal growth factor promotes plant growth.
Proceedings of the National Academy of Sciences of the United States of
America 77: 4836–4837.
29. Kato R, Nagayama E, Suzuki T, Uchida K, Shimomura TH, et al. (1993)
Promotion of plant cell division by an epidermal growth factor. Plant and Cell
Physiology 34: 789.
30. Bergman M (2002) Can saliva from moose, Alces alces, affect growth responses in
the sallow, Salix caprea? Oikos 96: 164–168.
31. Rooke T (2003) Growth responses of a woody species to clipping and goat saliva.
African Journal of Ecology 41.
32. Strauss SY, Agrawal AA (1999) The ecology and evolution of plant tolerance to
herbivory. Trends in Ecology & Evolution 14: 179–185.
33. Murphy JS, Briske DD (1992) Regulation of tilleri ng by apical dominance:
Chronology, interpretive value, and current perspectives. Journal of Range
Management 45: 419–429.
34. Zhang Z, Wang SP, Jiang GM, Patton B, Nyren P (2007) Responses of Artemisia
frigida Willd. (Compositae) and Leymus chinensis (Trin.) Tzvel. (Poaceae) to sheep
saliva. Journal of Arid Environments 70: 111–119.
35. McNaughton SJ (1983) Compensatory plant growth as a response to herbivory.
Oikos 40: 329–336.
36. Briske DD (1991) Developmental morphology and physiology of grasses. In:
Heitschmidt RK, Stuth JW, eds. Grazing management An ecological
perspective. Portland, Oregon USA: Timber Press. pp 85–108.
37. Tiffin P (2000) Mechanisms of tolerance to herbivore damage: What do we
know? Evolutionary Ecology 14: 523–536.
38. Tuomi J, Nilsson P, Astrom M (1994) Plant compensatory responses: bud
dormancy as an adaptation to herbivory. Ecology 75: 1429–1436.
39. Huhta AP, Lennartsson T, Tuomi J, Rautio P, Laine K (2000) Tolerance of
Gentianella campestris in relation to damage intensity: An interplay between apical
dominance and herbivory. Evolutionary Ecology 14: 373–392.
40. Wang DL, Ba L (2008) Ecology of meadow steppe in northeast China.
Rangeland Journal 30: 247–254.
41. Wang L, Wang D, He Z, Liu G, Hodgkinson KC (2010) Mechanisms linking
plant species richness to foraging of a large herbivore. Journal Of Applied
Ecology 47: 868–875.
42. Wang L, Wang D, Bai Y, Jiang G, Liu J, et al. (2010) Spatial distributions of
multiple plant species affect herbivore foraging selectivity. Oikos 119: 401–408.
43. Wang L, Wang D, Bai Y, Huang Y, Fan M, et al. (2010) Spatially complex
neighboring relationships among grassland plant species as an effective
mechanism of defense against herbivory. Oecologia 164: 193–200.
44. Teng X, Ba L, Wang D, Wang L, Liu J (2010) Growth responses of Leymus
chinensis (Trin.) Tzvelev to sheep saliva after defoliation. Rangeland Journal 32:
419–426.
45. Shi DC, Wang DL (2005) Effects of various salt-alkaline mixed stresses on
Aneurolepidium chinense (Trin.) Kitag. Plant and Soil 271: 15–26.
46. Ba L, Wang DL, Hodgkinson KC, Xiao NZ (2006) Competitive relationships
between two contrasting but coexisting grasses. Plant Ecology 183: 19–26.
47. Lothier J, Lasseur B, Le Roy K, Van Laere A, Prud’homme MP, et al. (2007)
Cloning, gene mapping, and functional analysis of a fructan 1-exohydrolase (1-
FEH) from Lolium perenne implicated in fructan synthesis rather than in fructan
mobilization. Journal of Experimental Botany 58: 1969–1983.
48. Olson BE, Richards JH (1988) Tussock regrowth after grazing: Intercalary
meristem and axillary bud activity of tillers of Agropyron desertorum. Oikos 51:
374–382.
49. Jewiss OR (1972) Tillering in grasses-its significance and control. Grass and
Forage Science 27: 65–82.
50. Flemmer AC, Busso CA, Fernandez OA (2002) Bud viability in perennial
grasses: Water stress and defoliation effects. Journal of Range Management 55:
150–163.
51. Belsky AJ (1986) Does herbivory benefit plants? A review of the evidence.
American Naturalist 127: 870.
52. McNaughton SJ, Wallace LL, Coughenour MB (198 3) Plant adaptation in an
ecosystem context: effects of defoliation, nitrogen, and water on growth of an
African C4 sedge. Ecology 64: 307–318.
53. Ongaro V, Leyser O (2008) Hormonal control of shoot branching. Journal of
Experimental Botany 59: 67–74.
54. Frazier WA, Boyd LF, Pulliam MW, Szutowicz A, Bradshaw RA (1974)
Properties and specificity of binding sites for
125
I-nerve growth factor in
embryonic heart and brain. Journal of Biological Chemistry 249: 5918–5923.
55. Harris P (1974) A possible explanation of plant yield increases following insect
damage. Agro-ecosystems 1: 219–225.
56. Wilson JB (1988) A review of evidence on the control of shoot: Root ratio, in
relation to models. Annals of Botany 61: 433–449.
57. Rosenthal JP, Welter SC (1995) Tolerance to herbivory by a stemboring
caterpillar in architecturally distinct maizes and wild relatives. Oecologia (Berlin)
102: 146–155.
58. Tomlin son KW, O’Connor TG (2004) Control of tiller recruitment in
bunchgrasses: Uniting physiology and ecology. Functional Ecology 18: 489–496.
59. Schlichting CD (1986) The evolution of phenotypic plasticity in plants. Annual
Reviews in Ecology and Systematics 17: 667–693.
Plant Response to Animal Saliva
PLoS ONE | www.plosone.org 8 January 2012 | Volume 7 | Issue 1 | e29259
