Field experimental evidence that grazers mediate transition between microalgal and seagrass dominance

Abstract

We tested the relative effects of nutrient loading, reduced predation, and reduced grazing on eelgrass
community dynamics in Chesapeake Bay and found evidence supporting the ‘‘mutualistic mesograzer model’’ in which small invertebrate grazers control accumulation of epiphytic algae, buffer eutrophication effects, and thus facilitate seagrass dominance. Experimental reduction of crustacean grazers in the field stimulated a nearly sixfold increase in epiphytic algae, and reduced seagrass biomass by 65% compared to controls with grazers. Nutrient fertilization generally had much weaker effects, but an interaction with mesograzers was key in changing the sign of fertilization effects on the system: aboveground eelgrass biomass was reduced by fertilization under reduced grazing, but increased by fertilization under ambient grazing. When protected from predators in field cages, these mesograzers limited epiphyte blooms even with nutrient enrichment, and nutrients instead enhanced grazer secondary production. Crustacean mesograzers play a key role in maintaining macrophyte (seagrass) dominance in Chesapeake Bay, in buffering eelgrass against eutrophication, and in efficiently transferring nitrogen to higher
trophic levels. Yet, these crustacean grazers are also highly sensitive to predator abundance. Reducing nutrient pollution alone is unlikely to restore seagrass meadows where alterations to food webs have reduced populations of algae-feeding mesograzers. Integration of both water quality and fishery management will be more effective in restoring and maintaining healthy coastal ecosystems.

Full text

Field experimental evidence that grazers mediate transition between microalgal and
seagrass dominance
Pamela L. Reynolds,
a,*
J. Paul Richardson, and J. Emmett Duffy
Virginia Institute of Marine Science, College of William & Mary, Gloucester Point, Virginia
Abstract
We tested the relative effects of nutrient loading, reduced predation, and reduced grazing on eelgrass
community dynamics in Chesapeake Bay and found evidence supporting the ‘‘mutualistic mesograzer model’’ in
which small invertebrate grazers control accumulation of epiphytic algae, buffer eutrophication effects, and thus
facilitate seagrass dominance. Experimental reduction of crustacean grazers in the field stimulated a nearly sixfold
increase in epiphytic algae, and reduced seagrass biomass by 65%compared to controls with grazers. Nutrient
fertilization generally had much weaker effects, but an interaction with mesograzers was key in changing the sign
of fertilization effects on the system: aboveground eelgrass biomass was reduced by fertilization under reduced
grazing, but increased by fertilization under ambient grazing. When protected from predators in field cages, these
mesograzers limited epiphyte blooms even with nutrient enrichment, and nutrients instead enhanced grazer
secondary production. Crustacean mesograzers play a key role in maintaining macrophyte (seagrass) dominance
in Chesapeake Bay, in buffering eelgrass against eutrophication, and in efficiently transferring nitrogen to higher
trophic levels. Yet, these crustacean grazers are also highly sensitive to predator abundance. Reducing nutrient
pollution alone is unlikely to restore seagrass meadows where alterations to food webs have reduced populations
of algae-feeding mesograzers. Integration of both water quality and fishery management will be more effective in
restoring and maintaining healthy coastal ecosystems.
A fundamental challenge in ecology is understanding
and predicting how changes in the abiotic and biotic
environment influence the structure and function of
ecosystem processes (Gruner et al. 2008). Understanding
the interplay of bottom-up and top-down processes,
including the supply of nutrients and consumer pressure,
is critical given ongoing perturbations to environmental
condition and food-web topology (Duffy 2003; Byrnes et
al. 2007). The importance of quantifying the synergistic
effects of bottom-up and top-down control is well
illustrated by seagrass ecosystems, which are highly
threatened by both the bottom-up effects of eutrophication
and the top-down effects of overfishing, among other
human activities. Seagrasses provide important ecologic
and economic services from provision of nursery habitat to
shoreline protection against erosion and storm events to
carbon sequestration (Costanza et al. 1997), but seagrasses
worldwide have declined dramatically over recent decades
(Orth et al. 2006b; Waycott et al. 2009).
Historically, responses to seagrass declines have focused
on eutrophication, associated high turbidity, and blooms of
epiphytic and planktonic algae (bottom-up control), which
compete with seagrasses for light and other resources
(McGlathery 1995; Hauxwell et al. 2001). Although good
water quality is clearly important for seagrass vigor (Orth
et al. 2006a), growing evidence also supports an important
role for top-down control in seagrass systems (reviewed by
Hughes et al. 2004; Baden et al. 2010; Duffy et al. 2013),
with important implications for the extensive efforts and
expense devoted to seagrass management and restoration
worldwide. In particular, the small invertebrate herbivores
that dominate many coastal and estuarine systems often
appear to play key roles in shifts between alternative
community states dominated by macrophytes (e.g., sea-
grasses) and microalgae. These small crustacean and
gastropod mesograzers are thought to function as a critical
link in coastal food webs similarly to zooplankton in
pelagic systems, and they can shift primary producer
dominance by mediating trophic cascades (Duffy and
Hay 2000; Davenport and Anderson 2007). Mesograzers
are often the dominant herbivores in temperate seagrass
systems (Cebrian 1999; Valentine and Duffy 2006) and,
while they have been observed in select cases to act as pests
and inhibit their hosts through direct consumption of
seagrass tissue (Zimmerman et al. 2010; Reynolds et al.
2012), in most instances they feed preferentially and heavily
on ephemeral macroalgae and microalgae (reviewed by
Jernakoff et al. 1996; Valentine and Duffy 2006).
Because algae are typically competitively superior to
seagrasses, especially under high-nutrient conditions, me-
sograzers may play a key role in preventing overgrowth of
seagrasses by algae and therefore act as mutualists with
their hosts, promoting seagrass growth and buffering them
against algal overgrowth resulting from nutrient pollution.
This ‘‘mutualistic mesograzer’’ model (Duffy et al. 2013)
was originally based on natural history observations and
lab experiments (Orth and Van Montfrans 1984) and is
supported by growing field evidence (Baden et al. 2010;
Cook et al. 2011; Whalen et al. 2013), although there are
exceptions (Lewis and Anderson 2012).
The important role of grazing in seagrass systems raises
the question of what factors control grazers and their algal
resources. Invertebrate grazers, particularly crustaceans,
* Corresponding author: plreynolds@ucdavis.edu
a
Present address: Department of Environmental Science and
Policy, University of California, Davis, California
Limnol. Oceanogr., 59(3), 2014, 1053–1064
E2014, by the Association for the Sciences of Limnology and Oceanography, Inc.
doi:10.4319/lo.2014.59.3.1053
1053

are important prey for fishes (Edgar and Shaw 1995) and are
highly vulnerable to predation. Thus, disturbances to upper
levels of the food web, e.g., through fishing, could have
pervasive effects that ripple through food webs and ecosys-
tems (Heck and Valentine 2007). Recent evidence from the
Swedish west coast supports this hypothesis, showing declines
in top predators and concomitant increases in mesopredator
populations coinciding with reduced grazer abundances and
dramatic seagrass declines (Baden et al. 2012).
Although many lines of evidence now support a role for
cascading top-down effects in vegetated marine communi-
ties, few if any studies have unequivocally documented the
final links in the proposed mutualism, that is, from
mesograzer reduction to algal bloom to reduced seagrass
performance and biomass. It is also unclear how general
this model of mesograzer control of alternate vegetation
states is within estuaries (Duffy et al. 2013). In many
temperate estuaries, invertebrate grazers in seagrass beds
are consumed by a diverse suite of small predators
including decapod crustaceans and demersal fishes (Orth
et al. 1984; Teixeira and Musick 1994), most of which are
generalists capable of strongly controlling grazer popula-
tions with potential consequences for algal–seagrass
dynamics in this system. These small predators may be an
important link in seagrass systems, since fluctuations in
their populations and overall predation intensity may lead
to grazer and/or algal outbreaks (Baden et al. 2010;
Svensson et al. 2012). While nutrient loading has histori-
cally been attributed as the driver for shifts in seagrass to
algal dominance (Harlin 1993; Orth et al. 2006b; Bur-
kholder et al. 2007), trophic interactions are clearly also
important (Heck et al. 2000; Hughes et al. 2004). In the
presence of small invertebrate herbivores, fertilization
alone has shown little negative effect on seagrass growth
(reviewed by Hughes et al. 2004; Burkepile and Hay 2006;
Valentine and Duffy 2006), and thus the question remains:
can small invertebrate grazers mediate eutrophication
effects in coastal food webs?
Here we investigate the interactive influence of bottom-up
and top-down forcing on seagrass systems in Chesapeake
Bay, Virginia. Using a combination of cageless and tradi-
tional caging methods in the field, we evaluate the potential
role of small crustacean grazers in buffering this system from
nutrient-mediated algal blooms and the effect of small
predators (fish, shrimp, crabs) in regulating these communi-
ties. Specifically, we examined the effects of fertilization and
grazer and predator exclusion on epiphyte accumulation,
seagrass production, and grazer biomass in the field.
Methods
We conducted two experiments to evaluate the relative
importance of top-down effects of consumers and the
bottom-up influence of fertilization on the dynamics of
eelgrass (Zostera marina) communities in the field. Histor-
ically the role of mesograzers in realistic field situations has
been very difficult to address owing to the difficulty of
caging them (Connell 1974; Virnstein 1978; Miller and
Gaylord 2007). The first experiment used a novel cageless
method to exclude grazers (Poore et al. 2009), while the
second featured cages to exclude their predators. Addi-
tionally, a predation assay was conducted in the field to
assess top-down pressure on the most abundant mesograzer
taxa and to confirm hypothesized food-web relationships.
All experiments were conducted in a dense bed of Z. marina
on the southeast side of the Goodwin Islands National
Estuarine Research Reserve on the York River estuary near
the mouth of the Chesapeake Bay in Virginia (37u139N,
76u239W) at 0.5 m mean-low-water depth.
Mesograzer exclusion—To test the role of grazers and
nutrient additions on the seagrass community, we estab-
lished experimental plots in May of 2011 using cageless
methods (modified from Whalen et al. 2013). Plots were
framed by three polyvinyl chloride (PVC) poles forming a
0.5 m sided equilateral triangle and randomly assigned to
one of four experimental treatments in a factorial design.
For the nutrient addition treatment, plots received either
0 or 300 g of the commercial, slow-release (4 month)
Plantacote
TM
fertilizer (14 nitrogen, 14 phosphorus, 14
potassium) placed in fiberglass mesh (1 mm) bags attached
30 cm from the sediment to each plot pole, such that
fertilized plots received a total of 900 g and nonfertilized
plots received only empty bags (methods comparable to
Moksnes et al. 2008; Baden et al. 2010). Although the
fertilizer slowly leaches into the water column over a period
of 6–8 weeks, to ensure consistent delivery throughout the
experiment the bags were replaced with new fertilizer after
4 weeks. Prior work in the York River, Virginia, and
ongoing monitoring by the Chesapeake Bay National
Estuarine Research Reserve show pronounced fluctuations
in nutrients and phytoplankton in patterns that are not
indicative of chronic background eutrophication (Whalen
et al. 2013). Our fertilizer application increased nitrogen
content in the eelgrass leaves by over 20%(see Results),
simulating a chronic, eutrophic condition.
Experimental reduction of mesograzers was accom-
plished by fitting each pole in a plot with a plaster (Ortho
Plaster, 275-310 bar slow) block containing 10%carbaryl
(Bayer Crop Science) by weight, which reduces crustacean
grazer densities in the field (Whalen et al. 2013), attached
15 cm from the sediment. Carbaryl, a reversible acetylcho-
linesterase inhibitor widely used against arthropods in
homes, gardens, and aquaculture in the United States, has
had limited direct effects on nonarthropod taxa (such as
algae) in experimental studies (Carpenter and Lodge 1986;
Duffy and Hay 2000; Dumbauld et al. 2001). Supplemental
studies found the concentration used here effectively
reduced gammaridean amphipod densities in the field up
to 60 cm away from the block, with greatly diminished
effectiveness at greater distances, supporting the highly
localized effects of this treatment (Whalen et al. 2013).
Control plots received plaster blocks without carbaryl.
Blocks were prepared with a recipe modified from Poore
et al. (2009) and were replaced weekly throughout the
experiment. This cageless design allows experimental
exclusion of small crustacean grazers from seagrass in the
field without strong modification of flow, light, and other
parameters that can be compromised in traditional caging
experiments (Connell 1974; Virnstein 1978).
1054 Reynolds et al.

Table 1. Mesograzer exclusion experiment: Effects of experimental treatments (deterrent and nutrient additions) on mesograzer
biomass at 4 weeks, epiphyte (mg Chl aper gram of Z. marina) and epibiont load at 4 weeks, total seagrass biomass at 8 weeks, and Z.
marina production and tissue percent nitrogen at 4 weeks. Values significant at p,0.05 are listed in italic. df, degrees of freedom.
Response df Sum squares Fp
Total mesograzer biomass
Deterrent 1 2.256 7.036 0.012
Nutrients 1 0.001 0.001 0.972
Deterrent 3nutrients 1 0.016 0.050 0.824
Error 36 11.544
Epiphytes
Deterrent 1 1.754 10.836 0.002
Nutrients 1 0.001 0.006 0.940
Deterrent 3nutrients 1 0.396 2.449 0.126
Error 36 5.827
Epibionts
Deterrent 1 5.682 81.829
,
0.001
Nutrients 1 0.025 0.366 0.549
Deterrent 3nutrients 1 0.194 2.791 0.103
Error 36 2.500
Total seagrass biomass
Deterrent 1 0.78 43.410
,
0.001
Nutrients 1 0.032 1.760 0.193
Deterrent 3nutrients 1 0.044 2.383 0.131
Error 36 0.663
Z. marina biomass
Aboveground
Deterrent 1 0.421 13.218 0.001
Nutrients 1 0.058 1.830 0.185
Deterrent 3nutrients 1 0.146 4.584 0.039
Error 36 1.147
Belowground
Deterrent 1 0.789 5.059 0.031
Nutrients 1 0.000 0.001 0.973
Deterrent 3nutrients 1 0.005 0.033 0.858
Error 36 5.615
R. maritima biomass
Aboveground
Deterrent 1 1.447 7.172 0.011
Nutrients 1 0.081 0.340 0.531
Deterrent 3nutrients 1 0.290 1.437 0.238
Error 36 7.265
Belowground
Deterrent 1 1.040 7.309 0.010
Nutrients 1 0.427 2.998 0.092
Deterrent 3nutrients 1 0.569 3.996 0.053
Error 36 5.123
Zostera production
Deterrent 1 1.676 4.663 0.038
Nutrients 1 0.214 5.952 0.020
Deterrent 3nutrients 1 0.082 2.272 0.141
Error 34 1.222
Zostera tissue %N
Deterrent 1 0.015 3.911 0.056
Nutrients 1 0.018 4.780 0.035
Deterrent 3nutrients 1 0.003 0.719 0.402
Error 36 0.137
Grazers structure eelgrass communities 1055

Four weeks after treatment application, we collected Z.
marina shoots and associated algae and epifauna to test for
treatment effects. We quantified the mesograzer communi-
ty by collecting all macrophytes in approximately a 19 cm
diameter circle in the center of each plot using a 300 mm
mesh bag. Grazers were counted and sorted by species, as
well as by size class using a stack of nested sieves. We then
estimated total biomass and secondary production from
the sieve size class abundance data using taxon-specific
empirical equations (as in Edgar 1990). We then standard-
ized mesograzers by the total biomass (grams of dry mass)
of macrophytes collected per sample.
Additional collections of one Z. marina shoot per plot
were made to quantify epiphytic algae (chlorophyll aper
dry mass [g] of the scraped leaves), and four Z. marina
shoots per plot to quantify the total mass of epibiota
(pooled dry mass of all material scraped from the four
leaves standardized by total leaf dry mass). We used whole
shoots instead of single leaves to examine epiphyte and
epibiont loading as leaf age can influence fouling accumu-
lation. These collections were repeated after 8 weeks. After
8 weeks we also harvested all remaining seagrass per plot.
This material was separated into species (Z. marina, Ruppia
maritima) and into aboveground vs. belowground tissue
and dried to assess differences in biomass across experi-
mental treatments.
We quantified treatment effects on the growth rate of the
Z. marina within the plots by punching at least five shoots
(just above their respective leaf sheaths) per plot 3 weeks
after the experiment was initiated. These shoots were
collected a week later (timepoint week 4) and taken back to
the laboratory, where we then measured the linear
extension of the leaves and calculated the rate of dry mass
accumulation (g d
21
; Short and Duarte 2001). Estimated
growth rates from all shoots in a plot were pooled to obtain
a plot-level mean, which we used to examine treatment
effects on eelgrass growth rate (mm new growth extension
3(Z. marina mg 3mm
21
)3duration
21
). To assess
treatment effects on Z. marina leaf stoichiometry, at the
4 week time point we collected five shoots from the edge of
each plot and removed the youngest leaf from each shoot.
A 3 cm piece of each of these leaves was rinsed with
deionized water, and this tissue was then pooled by plot,
dried, ground, and processed on a carbon–hydrogen–
nitrogen (CHN) analyzer at the Virginia Institute of
Marine Science (VIMS) to assess treatment differences in
leaf tissue nitrogen content. The leaf tissue nitrogen served
as an integrated metric to assess nutrient availability for the
eelgrass (e.g., fertilization treatment effectiveness at elevat-
ing water column nitrogen) during the field experiment
(Burkholder et al. 2007). Epiphyte tissue was not available
for CHN analysis, although owing to its faster turnover
rates we do not necessarily expect to have been able to
strongly detect fertilization effects on microalgal stoichi-
ometry.
We tested deterrent and nutrient treatment effects on
plant and epifaunal biomass, epiphyte load, and Z. marina
growth and leaf tissue nitrogen content using two-way
Fig. 1. Effects in mesograzer exclusion experiment of deterrent application (control, DØ;
addition, D+) and nutrient additions (N+) or control (NØ) treatments on total mesograzer,
amphipod, isopod, and gastropod biomass per gram of macrophyte and epiphyte load (mgChlaper
gram of Z. marina) after 4 weeks, and final seagrass dry mass after 8 weeks in experimental field plots.
1056 Reynolds et al.

ANOVAs in R (R Development Core Team 2013, version
2.15.2) with type III sums of squares. To graphically
examine changes in the mesograzer community across
experimental treatments, we used nonmetric multidimen-
sional scaling (NMDS) based on Bray–Curtis distance with
the metaMDS function in the vegan package in R. The
results were plotted in two dimensions, and the envfit
procedure in vegan was used to overlay species vectors.
Direct effects of chemical deterrent—To confirm whether
the experimental deterrent treatment may have had direct
effects on the seagrasses in the mesograzer exclusion
experiment, we conducted a mesocosm experiment at
VIMS factorially manipulating seagrass species (Z. marina,
R. maritima) and deterrent (none, plaster only, plaster +
carbaryl) treatments (n58). Seagrasses were collected
from Allen’s Island, York River, Virginia, in October 2011
and transported back to the lab on ice where they were
gently defaunated by hand and rinsed in freshwater and salt
water to ensure removal of all grazers. Mesocosms
(18 liters) were randomly placed in tanks (blocking factor),
assigned to one of the four treatments, and filled with
4.5 liters of prepared sediment (30%mud, 70%sand
mixture). Mesocosms then received either 12 63gZ.
marina or 13 63gR. maritima and either no block or a 50 g
block composed of plaster only or plaster prepared with
10%carbaryl (deterrent treatment). Mesocosms received
pulsed dumps approximately every 2 min of 150 mm filtered
seawater from the adjacent York River and were shaded to
approximate light conditions in the field. Three Z. marina
shoots per mesocosm were punched at the leaf sheath to
quantify growth rate. After 17 d we harvested all seagrass
and scraped off all epibionts, which were dried and weighed
to quantify epiphyte load. Barnacles were removed and
dried separately to test deterrent treatment effects on this
fouling invertebrate. After scraping, we then lightly rinsed
and weighed all seagrass shoots. Responses were analyzed
separately for the two seagrass species with one-way
blocked ANOVAs in R.
Predation assay—To assess the selectivity of predation in
the field, we employed a predation assay (Manyak et al.
2013) examining differences among mesograzer taxa in
vulnerability to predators in the field. Although gammar-
idean amphipod, isopod, and gastropod grazers are all
consumed by larger invertebrates and fishes, predator
selection and preferences may vary across taxa with
potential effects on epiphyte–seagrass interactions depend-
ing on which grazer taxa, and their relative grazing effects,
are more susceptible to predation (Best and Stachowicz
2012; Eklo¨ f et al. 2012). To test predator effects across
grazer taxa in the field, in July 2012 we used super glue to
attach one mesograzer (Ampithoid amphipod, isopod
Erichsonella attenuata, or gastropod Bittium varium), or a
standard prey type (10 mm piece of freeze-dried shrimp), or
a control prey mimic (10 mm piece of fuzzy pipe cleaner) to
Fig. 2. Effects of nutrient addition (control, NØ; addition, N+) and mesograzer deterrent application
(D+) on mesograzer community composition after 4 weeks in the mesograzer exclusion experiment,
illustrated via NMDS. Grazer taxa included amphipods Ampithoe longimana, Ampithoe valida, Caprella
equilibria, Caprella penantis, Corophium sp., Cymadusa compta, Elasmopus levis, Gammarus mucronatus,
Microprotopus raneyi, Paracaprella tenuis; isopod Edotea triloba; gastropods Bittium varium, Crepidula sp.
Grazers structure eelgrass communities 1057

a standardized location on a leaf of a shoot of Z. marina.
Individual grazers were collected within their most abun-
dant size class (5–15 mm) and represent the most common
mesograzer species at the field site. The shoot was then tied
to a clear acrylic rod for deployment in the field such that
the shoot was oriented upright in a natural orientation.
Rods were deployed at low tide and collected 24 h later. As
nearly all mimics remained attached, we inferred that
missing prey were likely eaten and not absent due to a loss
of glue adhesion. Prey presence vs. absence data were
analyzed with logistic regression in R, which took into
account baseline (control) losses.
Predator exclusion—To explore top-down effects of
predators on mesograzer and cascading effects on primary
producers, we conducted a 3 week field caging experiment
manipulating predator access and nutrient addition in plots
established in July 2012. Thirty plots were randomly
assigned in a two-way factorial design to experimental
nutrient addition (two levels: 0 or 300 g Plantacote
TM
) and
predator exclusion (three levels: open plot, cage control, full
cage). Cages were constructed using a frame of PVC (40 3
40 345 cm length, width, height) wrapped in nylon mesh
(0.79 mm diameter holes), which allowed access to the cage
by grazers but excluded the larger predatory fishes and
invertebrates. All plots were established along a seagrass
patch edge and were marked with two PVC poles; cages were
secured to these poles with bungee cords. Cages were filled
with approximately 5 liters of coarsely defaunated mud and
Fig. 3. Effect in mesograzer exclusion experiment of deterrent application (D+) and nutrient
additions (N+)onZ. marina and R. maritima aboveground (shoots) and belowground (roots,
rhizomes) biomass after 4 weeks in the mesograzer exclusion experiment.
Fig. 4. Effects in mesograzer exclusion experiment of deter-
rent application (D+) and nutrient additions (N+) treatments on
the percentage nitrogen in new growth Z. marina leaf tissue, and
on Z. marina growth rate after 4 weeks in the field.
1058 Reynolds et al.

sand to provide substrate and assist in anchoring the cage.
Open plots received a square of PVC placed on the sediment
and secured to the marking poles. A circle of Vexar (30 cm
diameter), to which 24 shoots of live Z. marina were cable
tied at natural densities (initial wet mass of ,35 g per plot),
was attached to the bottom PVC square of each plot. Z.
marina shoots were collected with their associated rhizomes
and root hairs and were gently rinsed in seawater to remove
any fauna and debris prior to attachment. After the eelgrass
was added, all plots were then stocked with natural
assemblages of grazers (,75 amphipods and ,50
gastropods) collected by conducting two, 2 m long dipnet
(13 314.5 cm) sweeps within the center of the seagrass bed
adjacent to each plot. Nutrients were placed in mesh bags as
in the mesograzer exclusion experiment and attached to one
of the poles next to the cage. Plots were monitored and cages
cleaned weekly throughout the experiment.
After 3 weeks, we enclosed each Vexar circle with its
attached Z. marina and associated epifauna and predators
in a mesh bag and transported them to the lab where we
quantified the final dry mass of Z. marina, abundance and
biomass of associated grazers by gross taxonomic group
(gastropods, amphipods, and isopods), and identity and
abundance of predators. Epiphytic algal load was quanti-
fied from one separately collected shoot per plot as in the
mesograzer exclusion experiment. Since previous experi-
ments have demonstrated that cages can introduce unin-
tended artifacts due to reductions in light, water flow, and
other factors (Connell 1974; Virnstein 1978), our interest
Table 2. ANOVAs of treatment effect (control, plaster only,
plaster +carbaryl) on given responses in experimental mesocosms.
Response df Sum squares Fp
Z. marina growth
Treatment 2 34.738 2.050 0.158
Blocking 1 18.716 2.209 0.155
Treatment 3blocking 2 4.641 0.274 0.764
Error 18 152.498
Z. marina growth rate
Treatment 2 0.037 0.771 0.479
Blocking 1 0.005 0.194 0.666
Treatment 3blocking 2 0.059 1.221 0.321
Error 16 0.387
R. maritima growth
Treatment 2 27.69 0.352 0.708
Blocking 1 161.82 4.118 0.058
Treatment 3blocking 2 51.34 0.653 0.532
Error 18 707.39
Barnacles on Z. marina
Treatment 2 0.001 0.051 0.950
Blocking 1 0.002 0.436 0.518
Treatment 3blocking 2 0.022 2.056 0.157
Error 18 0.096
Barnacles on R. maritima
Treatment 2 0.030 2.676 0.096
Blocking 1 0.000 0.001 0.970
Treatment 3blocking 2 0.012 1.048 0.371
Error 18 0.102
Fig. 5. Effects of plaster and deterrent (10%carbaryl) on seagrass growth and barnacle accumulation in experimental mesocosms
after 17 d.
Grazers structure eelgrass communities 1059

was in contrasts specifically between the partial and full
caging treatments, and thus analyses were performed as in
Hindell et al. (2001) using a priori contrasts comparing the
partial to the full treatments. Two-way ANOVAs in R were
used to test for effects of nutrient addition (6), cage
treatment (partial, full), and their interactions on epiphyte
load and final Z. marina biomass and mesograzer wet mass
in the predator exclusion experiment.
Results
Mesograzer exclusion—The dilute, slowly dissolving
chemical deterrent reduced invertebrate mesograzer bio-
mass by 76%, and this mesograzer reduction cascaded to
increase epiphytic algae (chlorophyll a[Chl a]) by 590%on
average after 4 weeks, which in turn reduced seagrass
biomass by 65%after 8 weeks (Table 1; Fig. 1). The total
biomass of epibiota on the eelgrass was positively
correlated with chlorophyll a(p,0.0001, R
2
50.47),
was higher in the plots with reduced mesograzer biomass,
and was thus likely largely composed of epiphytic algae.
The chemical deterrent specifically reduced crustaceans,
with little to no effect on gastropods (Fig. 1). The
deterrent, but not the nutrient addition, substantially
shifted mesograzer community composition after 4 weeks
as a result of greatly reduced amphipod abundances
(Fig. 2). Nutrient additions had no effect on either epiphyte
load or mesograzer abundance in this experiment (Table 1;
Fig. 1).
Both eelgrass and widgeongrass (R. maritima) declined
on average in plots exposed to the deterrent (Table 1;
Fig. 3). This change was evident in both the aboveground
(leaves) and belowground (roots and rhizomes) compo-
nents of both seagrass species after 8 weeks in the field.
Deterrent (D) and nutrient (N) treatments interactively
affected Z. marina aboveground biomass, since greater leaf
biomass was observed in fertilized plots with the intact
ambient mesograzer community (DØ N+treatments).
Fig. 6. Prey survivorship by mesograzer type and seagrass
(habitat) type in predation assays.
Table 3. Predator exclusion experiment: Results of a priori contrasts of experimental treatments (caging, nutrient addition) on final mesograzer
biomass by taxa, epiphyte load (Chl amgpergramofZ. marina), and Z. marina dry mass after 3 weeks. Standard error (SE); Pr(.|t|) ,0.05 are
in italics.
Response Estimate SE tPr(.|t|)
Crustacean grazer biomass
Open plot vs. caging 21.459 0.591 22.467 0.022
Partial vs. full cage 20.887 0.348 22.546 0.018
Nutrient addition vs. no addition 20.894 0.422 22.116 0.045
Nutrients 3caging 20.177 0.348 20.508 0.616
Gastropod grazer biomass
Open plot vs. caging 1.131 0.408 2.770 0.011
Partial vs. full cage 0.911 0.241 3.786
,
0.001
Nutrient addition vs. no addition 0.060 0.292 0.205 0.839
Nutrients 3caging 0.163 0.241 0.678 0.505
Total mesograzer biomass
Open plot vs. caging 21.040 0.537 21.935 0.065
Partial vs. full cage 20.674 0.317 22.130 0.044
Nutrient addition vs. no addition 20.699 0.384 21.822 0.082
Nutrients 3caging 20.078 0.317 20.247 0.807
Epiphytes
Open plot vs. caging 0.907 0.257 3.526 0.002
Partial vs. full caging 0.209 0.154 1.353 0.190
Nutrient addition vs. no addition 20.004 0.186 20.021 0.983
Nutrients 3caging 0.075 0.154 0.487 0.631
Z. marina dry mass (g)
Open plot vs. caging 1.162 0.426 2.728 0.012
Partial vs. full caging 0.141 0.251 0.562 0.579
Nutrient addition vs. no addition 0.304 0.304 0.999 0.328
Nutrients 3caging 20.012 0.251 20.047 0.963
1060 Reynolds et al.

While fertilization promoted Z. marina production, meso-
grazer exclusion had the opposite effect (Fig. 4). Nitrogen
content of new Z. marina tissue was 9%higher in fertilized
plots and correlated with the increased growth rate in those
treatments (Fig. 4).
Direct effects of chemical deterrent—Z. marina and R.
maritima seagrasses grew by 9.7 and 7.6 g in wet mass, on
average, across all mesocosms during the experimental test
of potential direct effects of the chemical deterrent
treatment. No treatment (control, plaster only, plaster +
deterrent), blocking, or blocking by treatment interaction
effects were observed on the growth of Z. marina or R.
maritima, the growth rate of Z. marina, or the accumula-
tion of barnacles on each seagrass type (Table 2; Fig. 5).
Predation assay—Prey survivorship differed strongly
among prey taxa (amphipod, isopod, gastropod) and
seagrass habitats (Z. marina,R. maritima;x
2
511.42,
p50.010). Gammaridean amphipods were completely
removed by predators, whereas gastropods were largely
untouched (Fig. 6). Losses due to abrasion or weakened
glue adhesion in the field (measured as losses of the mimic)
were minimal in the eelgrass habitat and not observed at all
in the widgeongrass.
Predator exclusion—Few predators were recorded in the
full cages at the end of the experiment compared with
the partial cages (F
1,28
52.234, p50.018), confirming that
the experiment reduced predator pressure in the full cage
treatments. Predator exclosure increased crustacean but
reduced gastropod biomass (a priori contrast of partial vs.
full cage, Table 3; Fig. 7). Caged mesograzer communities
were dominated by gammaridean amphipods that are
known to be strong algal grazers (e.g., Ampithoe longimana,
Cymadusa compta,Gammarus mucronatus, Fig. 8). Nutri-
ent additions increased crustacean biomass but had no
effect on gastropod biomass (Table 3; Fig. 7). Neither
predator exclusion nor nutrient additions affected epiphyte
load (Chl aper gram of Z. marina) or the final mass of Z.
marina (a priori contrasts), although we did observe an
effect of caging itself (Table 3; Fig. 7).
Discussion
Our results provide direct field experimental confirma-
tions of the mutualistic mesograzer hypothesis, i.e., that
mesograzers promote seagrass dominance by cropping
algal competitors that otherwise reduce seagrass perfor-
mance (Orth et al. 1984; Duffy et al. 2013). Specifically,
experimental reduction of crustacean grazers stimulated a
nearly sixfold increase in epiphytic algae in the field, which
in turn strongly reduced seagrass growth and biomass (by
65%compared with controls). In general, we found
stronger evidence of top-down than of bottom-up control
in this system, although there were important effects of
fertilization on grazer production. In particular, the two
field experiments demonstrated that that grazers substan-
tially promoted eelgrass, nutrient loading enhanced bio-
mass of crustacean grazers but not of algae, and that
crustacean grazers were highly sensitive to the abundance
of predators. These results corroborate similar evidence of
mesograzer control of algae in laboratory and mesocosm
studies (reviewed by Jernakoff et al. 1996; Hughes et al.
2004; Valentine and Duffy 2006) as well as a growing
number of field experiments in eelgrass (Moksnes et al.
2008; Baden et al. 2010; Whalen et al. 2013) and other
seagrass systems (Cook et al. 2011; Myers and Heck 2013).
But few previous studies have made the final connection
from reduced grazing to ephemeral algal blooms to reduced
seagrass fitness.
Our results also support pioneering work in the Baltic
region, where experiments and time series also showed that
Fig. 7. Effects in predator exclusion experiment of caging
(open, partial, full) and nutrient addition (N+) on crustacean and
gastropod mesograzer biomass, epiphyte load, and Z. marina dry
mass after 3 weeks.
Grazers structure eelgrass communities 1061

mesograzers are a key link in eelgrass food webs, potentially
mediating the transition between macrophyte- and algal-
dominated systems (Moksnes et al. 2008; Baden et al. 2010,
2012). Our results thus confirm the emerging generalization
that temperate seagrass beds are much more sensitive than
historically assumed to perturbations to coastal food webs.
As in the Baltic region, however, the top-down control we
documented interacts in important ways with bottom-up
forcing. Specifically, in both systems, small crustacean
grazers play an important role in buffering seagrass against
eutrophication and improving seagrass health, as evidenced
by the interaction between fertilization treatment and
mesograzer presence on eelgrass biomass. In our study,
nutrient loading increased eelgrass biomass when mesogra-
zers were present but tended to reduce eelgrass biomass
when mesograzers were excluded; fertilization enhanced
eelgrass growth in both presence and absence of grazers in
our short-term assay. Similarly, experimentally enhanced
nutrient levels on the Swedish west coast resulted in blooms
of macroalgal mats and decreased growth of eelgrass in the
absence of mesograzers but had no effect under historically
high mesograzer densities (Baden et al. 2010). Likewise,
nutrient enrichment had no effect on shoalgrass biomass in a
recent field experiment (Myers and Heck 2013), although
there was a negative effect on leaf length in protected sites
where amphipod abundances were low. These interactions
between grazing and nutrient loading mirror many previous
experiments conducted in the laboratory and mesocosms
(Hughes et al. 2004), which showed that mesograzers
controlled the growth of ephemeral algae, even under
elevated nutrient conditions.
Seagrass ecology and the management strategies based
on it have historically focused almost exclusively on water
quality (specifically turbidity and nutrient loading). How-
ever, several of the studies discussed above from temperate
seagrass beds found that experimental fertilization had
little effect on epiphytic algae, whereas experimental
mesograzer reduction had stronger effects than fertilization
(Heck et al. 2000; Hughes et al. 2004; Spivak et al. 2009).
Nutrient limitation was supported in our study, however,
by the positive effect of nutrient enrichment on mesograzer
biomass in the predator exclusion study. This finding that
fertilization effects bypassed standing stock of primary
producers to elevate secondary production is similar to that
observed in a field caging experiment in the Baltic, where
ambient densities of grazers are high (Baden et al. 2010).
The key role of invertebrate grazers raises the question of
what controls their abundance. Both our study and
experiments in the Baltic region show that ambient
predator abundances exert strong top-down control on
grazers (Moksnes et al. 2008; Baden et al. 2010) and that
this pattern is overlain on, and interacts with, a base of
bottom-up control. Our field experiment confirmed previ-
ous mesocosm findings of a bottom-up influence on grazers
as nutrient loading was transmitted efficiently up the food web
to increase mesograzer biomass in mesocosm experiments in
Fig. 8. Effects of nutrient addition and cage type (open, partial, full) on mesograzer
community composition in the predator exclusion experiment after 3 weeks in the field, illustrated
via NMDS. Grazer taxa included species listed in Fig. 2 as well as amphipods Dulichiella
appendiculata,Lembos spinicarpus,Microdeutopus anomalus; isopod Paracerceis caudata,
gastropod Mitrella lunata, and an unidentified limpet.
1062 Reynolds et al.

Chesapeake Bay (Spivak et al. 2009) and field experiments in
Sweden (Moksnes et al. 2008). This bottom-up signal is
consistent with patterns in time-series data from our site
showing that correlations across trophic levels are more often
positive than negative (Douglass et al. 2010).
Although our data suggest that patterns of trophic
control are similar in eelgrass beds on both sides of the
North Atlantic, quite different processes can occur in other
seagrass systems (see review in Duffy et al. 2013), where,
for example, algal grazing by omnivorous fishes (Heck et
al. 2000) or destructive grazing of eelgrass tissue by
invertebrates (Zimmerman et al. 2010; Reynolds et al.
2012) has variable consequences for seagrasses when food
webs are perturbed. The relative strengths of top-down and
bottom-up control and their system-level consequences are
thus mediated by species- and system-specific factors,
including palatability of primary producers, food prefer-
ence and vulnerability of mesograzers (Best and Stachowicz
2012), omnivory of mesopredators (Heck and Valentine
2006), and differences in recruitment dynamics on different
trophic levels (Svensson et al. 2012). Similar to prior work
in Sweden (Baden et al. 2010), we found strong cascading
top-down control, likely because the most abundant
epifauna were effective algal grazers (primarily ampithoid
amphipods). These taxa were in turn highly vulnerable to
the ambient predator community, as evidenced by the
intense predation upon this group in field predation assays
(Fig. 6) as well as their dominance in communities in which
their predators were excluded (full field cages, Fig. 8).
Our results add growing support for the hypothesis that
changes in both nutrient regimes and coastal food-web
structure often interact to have a fundamental effect on
seagrass ecosystems. Reducing nutrient pollution alone is
unlikely to restore seagrass meadows if there have been
pervasive alterations to food webs that have resulted in
reduced populations of algal mesograzers. In these areas,
management of water quality would best be integrated with
fishery management to restore the abundance of large
predators and improve coastal ecosystems.
Acknowledgments
We thank the many technicians, students, and volunteers who
assisted with field and lab work, including R. Blake, S. Donadi, E.
Ferer, C. Fikes, M. Gyoerkoe, K. Jenkins, A. Kuwahara, J.
Lefcheck, K. Sobocinski, and M. Whalen. We also appreciate the
contributions of two anonymous reviewers and insights from the
Zostera Experimental Network. This work was supported by the
National Science Foundation grant 1031061 in Oceanography to
JED. This paper is Contribution No. 3354 of the Virginia Institute
of Marine Science, College of William & Mary.
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Received: 19 June 2013
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Amended: 12 February 2014
1064 Reynolds et al.