Thicker host tissues moderate light stress in a cnidarian endosymbiont

Abstract

The susceptibility of algal-cnidarian holobionts to environmental stress is dependent on attributes of both host and symbiont, but the role of the host is often unclear. We examined the influence of the host on symbiont light stress, comparing the photophysiology of the chlorophyte symbiont Elliptochloris marina in two species of sea anemones in the genus Anthopleura. After 3 months of acclimation in outdoor tanks, polyp photoprotective contraction behavior was similar between the two host species, but photochemical efficiency was 1.5 times higher in A. xanthogrammica than in A. elegantissima. Maximum relative electron transport rates, derived from rapid light curves, were 1.5 times higher in A. xanthogrammica than in A. elegantissima when symbionts were inside intact tissues, but were not significantly different between host species upon removal of outer (epidermis and mesoglea) tissue layers from symbiont-containing gastrodermal cells. Tissues of A. xanthogrammica were 1.8 times thicker than those of A. elegantissima, with outer tissue layers attenuating 1.6 times more light. We found no significant differences in light absorption properties per unit volume of tissue, confirming the direct effect of tissue thickness on light attenuation. The thicker tissues of A. xanthogrammica thus provide a favorable environment for E. marina - a relatively stress-susceptible symbiont - and may explain its higher prevalence and expanded range in A. xanthogrammica along the Pacific coast of North America. Our findings also support a photoprotective role for thicker host tissues in reef corals that has long been thought to influence variability in bleaching susceptibility among coral taxa.

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INTRODUCTION
Gathering sufficient light for photosynthesis is essential for
cnidarians such as corals and sea anemones that host algal
endosymbionts. At the same time, however, light and photosynthesis
can be sources of stress. Excess light, often in conjunction with
elevated temperature, can overburden symbiont photosynthetic
processes and lead to the disruptive loss of algal symbionts or their
pigments known as bleaching (Weis, 2008). Tolerance of light and
heat varies broadly among symbiont taxa, causing variation in
bleaching susceptibility and resulting in ecological zonation of
symbionts according to prevailing habitat conditions (Baker, 2003;
van Oppen et al., 2009). Symbiont identity has thus emerged as a
key determinant of both the distribution and stress susceptibility of
algal–cnidarian holobionts.
Meanwhile, several studies have concluded that the host plays
an equally important role in holobiont stress susceptibility (Bhagooli
and Hidaka, 2003; Goulet et al., 2005; Abrego et al., 2008; Fitt et
al., 2009). Hosts may differ in their ability to tolerate or limit
symbiont stress because of variation in, for example, antioxidant
enzymes, heat shock proteins, UV-absorbing compounds,
photoprotective pigments and behaviors, and heterotrophic capacity
(Salih et al., 2000; Brown et al., 2002; Grottoli et al., 2006; Fitt et
al., 2009; Baird et al., 2009). The resistance of certain scleractinian
coral taxa to bleaching is well known [the so-called ‘winners’ (Loya
et al., 2001; van Woesik et al., 2011)], yet the mechanisms behind
their resistance are not clearly understood, although hypotheses
include differences in acclimatization capacity, morphology-
mediated mass transfer efficiency and tissue thickness (Gates and
Edmunds, 1999; Loya et al., 2001). Many ‘winners’ have relatively
thick tissue, which has been speculated to provide bleaching
resistance through increased photoprotective capacity (Hoegh-
Guldberg, 1999; Loya et al., 2001). Photoprotection of symbionts
by host tissues is illustrated by studies showing increased light
sensitivity of isolated versus in hospite symbionts (Muller-Parker,
1984; Bhagooli and Hidaka, 2003; Goulet et al., 2005), as well as
by studies documenting considerable attenuation of light through
host tissues (Kühl et al., 1995; Magnusson et al., 2007; Kaniewska
et al., 2011). Although there is evidence of the importance of tissue
quality in photoprotection, including the presence of fluorescent and
non-fluorescent pigments (Salih et al., 2000; Dove et al., 2008) and
UV-absorbing mycosporine-like amino acids (MAAs) (Shick et al.,
1995), tissue thickness can differ substantially among host taxa
(Loya et al., 2001) and may be even more important to symbiont
photophysiology and ecology. To date, however, the potential for
thicker host tissues to mitigate symbiont stress has not been
evaluated.
Here, we were able to test the hypothesis that thicker host tissues
provide greater photoprotection and moderate symbiont stress in a
simple, tractable experimental system. We measured symbiont
photophysiology and host tissue properties in two closely related
(Geller and Walton, 2001) sympatric intertidal sea anemones
hosting a relatively stress-susceptible symbiont. Along the Pacific
coast of North America, the large solitary anemone Anthopleura
xanthogrammica and the smaller clonal anemone A. elegantissima
SUMMARY
The susceptibility of algal–cnidarian holobionts to environmental stress is dependent on attributes of both host and symbiont, but
the role of the host is often unclear. We examined the influence of the host on symbiont light stress, comparing the
photophysiology of the chlorophyte symbiont Elliptochloris marina in two species of sea anemones in the genus Anthopleura.
After 3months of acclimation in outdoor tanks, polyp photoprotective contraction behavior was similar between the two host
species, but photochemical efficiency was 1.5 times higher in A. xanthogrammica than in A. elegantissima. Maximum relative
electron transport rates, derived from rapid light curves, were 1.5 times higher in A. xanthogrammica than in A. elegantissima
when symbionts were inside intact tissues, but were not significantly different between host species upon removal of outer
(epidermis and mesoglea) tissue layers from symbiont-containing gastrodermal cells. Tissues of A. xanthogrammica were 1.8
times thicker than those of A. elegantissima, with outer tissue layers attenuating 1.6 times more light. We found no significant
differences in light absorption properties per unit volume of tissue, confirming the direct effect of tissue thickness on light
attenuation. The thicker tissues of A. xanthogrammica thus provide a favorable environment for E. marina – a relatively stress-
susceptible symbiont – and may explain its higher prevalence and expanded range in A. xanthogrammica along the Pacific coast
of North America. Our findings also support a photoprotective role for thicker host tissues in reef corals that has long been
thought to influence variability in bleaching susceptibility among coral taxa.
Key words: cnidarian, symbiosis, light stress, photosynthesis, photoprotection, bleaching.
Received 8 November 2011; Accepted 12 March 2012
The Journal of Experimental Biology 215, 2247-2254
© 2012. Published by The Company of Biologists Ltd
doi:10.1242/jeb.067991
RESEARCH ARTICLE
Thicker host tissues moderate light stress in a cnidarian endosymbiont
James L. Dimond1,*, Benjamin J. Holzman2and Brian L. Bingham3
1Shannon Point Marine Center, Western Washington University, Anacortes, WA, USA, 2Cascadia Community College, Bothell,
WA, USA and 3Department of Environmental Sciences, Western Washington University, Bellingham, WA, USA
*Author for correspondence (jdimond@gmail.com)
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host both dinoflagellate (Symbiodinium spp.) and chlorophyte
(Elliptochloris marina) symbionts. Relative to Symbiodinium
muscatinei,E. marina exhibits low tolerance of high light and
temperature, and is typically found in cooler, low-light habitats
(Bates, 2000; Verde and McCloskey, 2001; Verde and McCloskey,
2002; Secord and Muller-Parker, 2005; Dimond et al., 2011).
Interestingly, however, E. marina occurs approximately 6degrees
of latitude farther south and much higher in the intertidal zone in
A. xanthogrammica than in A. elegantissima (Secord and Augustine,
2000), indicating that host factors have an important effect on
symbiont distribution patterns. In this study, we show that E. marina
exhibits significantly less light stress in A. xanthogrammica than in
A. elegantissima, resulting from increased light attenuation through
A. xanthogrammica’s thicker tissues. Our results suggest that host
tissue thickness is both physiologically and ecologically relevant to
cnidarian symbioses, and support a long-standing yet previously
untested hypothesis about the role of thicker tissues in buffering
symbiont light stress.
MATERIALS AND METHODS
Experimental conditions
Sea anemones were collected at mean lower low water (±0.5m) at
two locations. Medium-sized Anthopleura xanthogrammica (Brandt
1835) (mean ± s.d. basal diameter7.7±0.8cm) were collected at
Slip Point, WA (48°15⬘51⬙N, 124°14⬘55⬙W), on 20 April 2011, and
large A. elegantissima (Brandt 1835) (mean basal
diameter3.4±0.5cm) were collected at Cone Island, WA
(48°35⬘34⬙N, 122°40⬘27⬙W), on 21 April 2011. At Shannon Point
Marine Center (48°30⬘32⬙N, 122°41⬘02⬙W), individuals of each
species were paired together in 10 independent outdoor tanks (69l
volume, 48cm water depth) each receiving flow-through seawater
at a rate of approximately 2lmin–1. Mean temperature of the tanks,
as measured by Hobo WaterTemp Pro dataloggers (Onset Computer,
Bourne, MA, USA) in two of them, was 11.3±0.5°C over the
experimental period. The anemones were fed a weekly ration of
one live mussel (Mytilus trossulus) proportional to anemone body
size. We verified the symbiont identity within anemones by viewing
symbionts extracted from an excised tentacle under light
microscopy; all anemones had >99% Elliptochloris marina Letsch
2009 both at the beginning and the end of the experiment.
Hemacytometer counts at the end of the experiment revealed
Symbiodinium muscatinei (LaJeunesse and Trench, 2000) at 0.1%
relative abundance in one A. xanthogrammica; S. muscatinei were
not detected in the other 19 anemones. Elliptochloris marina is easily
distinguished from Symbiodinium spp. due to its smaller size
(8–10m) and green color. Recent phylogenetic analysis of E.
marina confirmed its monophyly regardless of host species or
geographic location, and that analysis included specimens from
locales close to those sampled in our study (Letsch et al., 2009).
More recent and thorough population genetics analysis of rbcL and
ITS2 sequences has again found no significant geographic, seasonal
or host-species-specific population structure in E. marina (M.
Letsch, personal communication).
Sea anemone behavior and symbiont chlorophyll
fluorescence
Anemones were acclimated together in the outdoor tank system for
3months (April–July 2011) before collecting data on their
photobiology. This acclimation period is assumed to be sufficient
based on the results of Buchsbaum (Buchsbaum, 1968), who
monitored A. elegantissima hosting Symbiodinium after
transplantation to full sunlight and found stabilization of most animal
and algal pigments after only 7weeks. We first assessed anemone
photoprotective expansion/contraction behavior together with
symbiont chlorophyll fluorescence hourly between approximately
dawn and dusk over 2days. Photosynthetically active radiation
(PAR; 400–700nm) over the tanks was measured with a LI-COR
LI-190 2quantum sensor (LI-COR Biosciences, Lincoln, NE,
USA). The ranking scheme of Shick and Dykens (Shick and Dykens,
1984) was used to quantify anemone expansion/contraction behavior
(Fig.1A; 4fully expanded, 375% expanded, 250% expanded,
125% expanded, 0fully contracted). Given that Anthopleura spp.
are known for their photoprotective contraction behavior under high
The Journal of Experimental Biology 215 (13)
Time of day
6:00 12:00 18:00 0:00 6:00 12:00 18:00 0:00
Effective quantum yield (∆F/Fm⬘)
0
0.1
0.2
0.3
0.4
0.5
0.6
PAR (μmol quanta m–2 s–1)
0
500
1000
1500
2000
Expansion state
0
1
2
3
4
A. xanthogrammica
A. elegantissima
B
0
1
2
3
4
A
C
43 21 0
0
500
1000
1500
2000
Fig.1. Diel cycles of sea anemone expansion/contraction behavior and
symbiont photosystem II (PS II) quantum yield in Anthopleura
xanthogrammica and A. elegantissima. (A)Numerical ranking scheme of
anemone (A. elegantissima shown) photoprotective expansion/contraction
behavior based on Shick and Dykens (Shick and Dykens, 1984): 4fully
expanded, 375% expanded, 250% expanded, 125% expanded, 0fully
contracted. (B)Time series of anemone expansion/contraction behavior
(means ± s.e.m.) in outdoor flow-through seawater tanks over a 2-day
period in July 2011. There was no significant difference between species
(P0.69). (C)Time series of PS II quantum yield (F/Fm⬘; means ± s.e.m.)
of Elliptochloris marina in its two host anemones over the same period.
Pre-dawn values on the second day represent the dark-adapted maximum
quantum yield (Fv/Fm), which was significantly different between species
(P<0.001). Both time series are superimposed over ambient irradiance
levels (gray), shown on the right axes.
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2249Host tissues moderate symbiont stress
irradiance (Shick and Dykens, 1984), we deemed it necessary to
evaluate potential differences in behavior between species that could
influence the light dose experienced by different tissues, particularly
the oral disk and tentacles where most fluorescence data were taken.
The quantum yield of photosystem II (PS II) was measured using
a pulse-amplitude modulated (PAM) fluorometer (DIVING-PAM,
Heinz Walz, Effeltrich, Germany). The DIVING-PAM measures
PS II fluorescence by using a weak measuring light
(~0.15molquantam–2s–1) to assess the minimum fluorescence (Fo
in a dark-adapted state or Fin a light-adapted state), followed by
a 0.8s saturation pulse (>10,000molquantam–2s–1) that closes all
PS II reaction centers to assess maximum fluorescence (Fmin a
dark-adapted state or Fm⬘in a light-adapted state). Pre-dawn values
were used for assessment of the dark-adapted maximum quantum
yield (Fv/Fm[Fm–Fo]/Fm), whereas all other values represented the
effective quantum yield (F/Fm⬘[Fm⬘–F]/Fm⬘) under varying
degrees of illumination. The effective quantum yield reflects the
proportion of absorbed light energy that is used in photochemistry,
whereas the maximum quantum yield is the potential capacity of
PS II to use absorbed light energy for photochemistry when
chlorophyll reaction centers are in a relaxed, dark-adapted state.
Maximum quantum yield is a sensitive indicator of PS II light
history, stress and photoinhibition (Maxwell and Johnson, 2000).
During fluorescence measurements, the fluorometer probe was held
5mm from the anemone tissue surface, and care was taken to avoid
shading the tissue with the probe or eliciting anemone contraction
behavior by touching the tissue surface. When anemones were in
expanded posture, fluorescence readings were taken on the oral disk
adjacent to the first row of tentacles. Fluorescence readings of
contracted anemones were taken on the exposed column surface.
Rapid light curves
To evaluate the influence of the intact host tissue environment on
PS II function in E. marina, we performed rapid light curves (RLCs)
(Ralph and Gademann, 2005) with the DIVING-PAM on freshly
excised anemone tentacles as well as on symbionts extracted from
freshly excised tentacles the week following behavior and
fluorescence measurements in the experimental tanks. RLCs
measure F/Fm⬘at a range of light intensities emanating from the
fluorometer probe within a time span of 90s, and provide a test of
the ability of PS II reaction centers to tolerate short-term increases
in light intensity. RLCs were performed in a darkened room with
the fluorometer probe mounted 5mm over a large Petri dish filled
with seawater and maintained at 12°C. For each anemone, one to
four freshly excised intact tentacles were tested, and one to four
additional tentacles had their symbiont-containing gastrodermal cells
squeezed out of the severed end of the tentacles using a dental pick,
leaving behind the epidermal and mesogleal tissue. This extraction
method could be performed on a single tentacle within ~15s and
there was no evidence that cells were physiologically affected by
this procedure (see Results). The number of tentacles used was
dependent on their size and symbiont density, as it was necessary
to obtain sufficient symbionts for a strong fluorescence signal.
Samples were mounted on polycarbonate membrane filters
supported on glass slides and tested within 1–2min of being
transferred to the dark room. The RLC protocol involved eight
successive increases in PAR between 0 and 2700molquantam–2s–1
applied for 10s between each measurement of F/Fm⬘. This allowed
quantification of the relative electron transport rate (rETR) as
rETRF/Fm⬘⫻PAR. The maximum rETR (rETRmax) was
determined by fitting data with the hyperbolic tangent function
(Jassby and Platt, 1976; Ralph and Gademann, 2005).
Host tissue properties
To obtain relaxed tissue samples, three to four tentacles from each
anemone were excised at their base after reversible narcotization
with menthol crystals. Relaxed tentacles were fixed in 4%
paraformaldehyde in phosphate buffered saline for 24h. Light
attenuation by epidermal and mesogleal tissue layers was determined
by cutting a tentacle open lengthwise and removing gastrodermal
cells by gently scraping with a scalpel under a dissecting microscope,
then placing the strip of tentacle tissue over the PAR sensor (2.5mm
diameter) of the DIVING-PAM. The probe of the DIVING-PAM
was positioned 2cm directly over the PAR sensor and a constant
beam of actinic light (450molquantam–2s–1) was applied, with
PAR readings taken both with and without the tissue strip covering
the PAR sensor. The area of each tissue strip was determined by
photography under the dissecting microscope and subsequent image
analysis (ImageJ, National Institutes of Health, Bethesda, MD, USA)
after calibration with a stage micrometer. The tissue strip was then
frozen at –70°C for later determination of tissue light absorbance.
Through image analysis, we were able to confirm that very few
symbionts (gastrodermis) remained on the tissue strips (only
0.97±0.68% of tissue area still held gastrodermal cells). We were
also able to verify that mesoglea was not removed from these strips
during the dissection process; epidermis was instantly stripped from
mesoglea during the homogenization process, whereas the tougher
mesoglea initially remained intact until it eventually broke up and
became homogenized. We confirmed this by viewing partially
homogenized tissue strips under epifluorescence microscopy, noting
that the non-fluorescent mesoglea with no adherent epidermis
remained largely intact until further homogenization.
A second set of tentacles was embedded in optimum cutting
temperature compound (Tissue-Tek O.C.T. Compound, Sakura
Finetek, Flemingweg, The Netherlands) and frozen at –70°C for
later sectioning and measurement of tissue thickness. Frozen tentacle
blocks were sectioned at 20m using a rotary microtome (A0820,
AO Scientific Instruments, Buffalo, NY, USA) and tissue sections
were photographed under epifluorescence microscopy (Leitz DMR,
Leica Microsystems, Wetzlar, Germany) with a 450–490nm
excitation filter. The thickness of epidermal, mesogleal and
gastrodermal tissue layers was measured at the thickest part of each
section by image analysis after calibration with a stage micrometer.
The in vitro absorption spectrum of tentacle tissue was determined
by homogenizing the frozen epidermal and mesogleal strips
described above in 0.1moll–1 phosphate buffer with a motorized
Teflon tissue grinder. Following 24h under refrigeration, tissue
extracts were centrifuged at 10,000gfor 5min and read on an Agilent
8453A UV-Vis spectrophotometer (Agilent Technologies, Santa
Clara, CA, USA). Absorbance was normalized to epidermis volume
based on measurements of epidermis area and thickness as described
above.
Chlorophyll analysis
Chlorophyll content of E. marina was assessed by both
spectrophotometry and flow cytometry. For both analyses, symbiont
cell suspensions were obtained by homogenizing frozen (–70°C)
excised tentacles in filtered seawater with a motorized Teflon tissue
grinder. Spectrophotometry was performed according to methods
described by Dimond et al. (Dimond et al., 2011). Flow cytometry
was performed using a BD FACSCalibur instrument (BD
Biosciences, Franklin Lakes, NJ, USA) with a 488nm excitation
laser. Relative cell size was determined via the forward scatter (FSC)
detector, whereas chlorophyll fluorescence (FL3) was measured via
the 650nm long-pass detector. Acquisition was set to 10,000 cells
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accumulating in a pre-set gate on the FSC–FL3 plot, with logarithmic
amplification on both detectors. Histograms of FL3 and FSC were
modeled manually in Cyflogic 1.2 (CyFlo Ltd, Turku, Finland) to
obtain population means.
Photosynthetic efficiency after acclimation to low light
To evaluate photosynthetic efficiency in E. marina in the absence
of full sunlight and demonstrate that there were no inherent
differences in symbiont photophysiology between host species, we
measured Fv/Fmin anemones several months after our experiments
when sea anemones had become acclimated to an indoor, low-light
environment. In August 2011, the anemones were transferred to an
indoor flow-through seawater tank where they received natural light
through north-facing windows. Five months later, we measured
Fv/Fmin the anemones in the evening following 30min in complete
darkness. Irradiance levels at mid-day during this period were
approximately 30–50molquantam–2s–1.
Statistical analyses
Expansion/contraction behavior and PS II quantum yield of the two
anemone species over two diel cycles were evaluated with repeated-
measures ANOVA followed by tests of simple main effects. Paired
Student’s t-tests were used for all other comparisons except for Fv/Fm
of symbionts under low-light conditions, for which samples were
not paired in individual tanks and a standard Student’s t-test was
used. All data sets were homoscedastic except for epidermis
thickness, which was log-transformed prior to analysis to stabilize
variances. Statistical significance was determined at 0.05 and all
analyses were performed using SPSS Statistics 18 (IBM, Armonk,
NY, USA).
RESULTS
Host behavior and symbiont chlorophyll fluorescence
Over two diel cycles, we found no significant difference (F1,180.16,
P0.69) in expansion/contraction behavior of the two anemone
species (Fig.1B), indicating that anemone tissue surfaces received
similar light doses throughout the day. There were, however,
significant differences in PS II quantum yield (Fig.1C) between the
two anemone species for most but not all time periods, as indicated
by a significant time ⫻species interaction effect (F36,6482.93,
P<0.001). Simple main effects showed that although quantum yields
were often not significantly different between species during the
middle of the day, quantum yields in A. xanthogrammica were
significantly higher the majority (76%) of the time. Most
importantly, the pre-dawn maximum quantum yield (Fv/Fm) at
04:00h on day 2 was 1.5 times higher (P<0.001) in A.
xanthogrammica than in A. elegantissima (Fig.1C).
Rapid light curves
The rETRmax of symbionts in intact whole tentacles (Fig.2) was
significantly (1.5 times) higher in A. xanthogrammica (t2.5, d.f.9,
P0.03). In contrast, when symbionts were extruded from tentacles,
rETRmax values of symbionts from the two anemone species were
not significantly different (t1.3, d.f.9, P0.21). Thus, the presence
of intact epidermal and mesogleal tissue layers was largely
responsible for differences in rETRmax between anemone species,
suggesting that there were differences in the internal light
environment because of host-tissue properties. We were able to rule
out potential negative effects of the gastrodermal extrusion process
as initial measurements of F/Fm⬘, within minutes of tissue extrusion
and seconds before commencement of the RLC, showed no
significant difference (t1.4, d.f.19, P0.19) between symbionts
remaining in intact whole tentacles (0.198±0.077) and symbionts
extruded from epidermis and mesoglea (0.185±0.077).
Host tissue properties
Examination of host tissue properties revealed similar overall tissue
morphology of the two host species, including the presence of green
fluorescent protein in the epidermis (Fig.3A). However, all tissue
layers were considerably thicker in A. xanthogrammica than in A.
elegantissima (Fig.3A,B). Overall, tissues were 1.8 times thicker
in A. xanthogrammica with significant differences in epidermis
(t2.5, d.f.9, P0.03), mesoglea (t4.6, d.f.9, P0.001) and
gastrodermis (t5.6, d.f.9, P<0.001). Attenuation of PAR by strips
of tentacle epidermis and mesoglea was significantly (1.6 times)
greater in A. xanthogrammica than in A. elegantissima (t4.3, d.f.9,
P0.002; Fig.3C). The absorption spectra of tissue extracts (Fig.4)
showed slightly higher epidermis volume-specific absorption of A.
elegantissima tissue, but differences were not significant based on
overlap of 95% confidence intervals.
Chlorophyll analysis
Spectrophotometric analysis of cellular chlorophyll content in E.
marina indicated that symbionts from A. elegantissima had
significantly more chlorophyll a and b than those from A.
xanthogrammica, but similar ratios of chlorophyll a to chlorophyll
b (Table1). Flow cytometric analysis also verified that symbionts
from A. elegantissima had significantly higher chlorophyll content,
as indicated by higher FL3 red fluorescence. However, FL3
fluorescence normalized to relative cell size (FSC) showed no
significant difference between symbionts from the two host species.
There was a trend towards higher mean cell size (FSC) of symbionts
from A. elegantissima, but this was not significant (Table1).
Photosynthetic efficiency after acclimation to low-light
After 5months in the indoor tank under low-light conditions, Fv/Fm
of the symbionts in the two hosts was indistinguishable (A.
xanthogrammica0.708±0.017, A. elegantissima0.696±0.019;
t1.5, d.f.18, P0.16), indicating no inherent host-specific
differences in the photophysiology of the symbionts in the absence
of strong light. These Fv/Fmvalues were similar to those obtained
during the summer in other non-experimental anemones of both
species hosting E. marina in indoor seawater tables exposed to
natural sunlight.
The Journal of Experimental Biology 215 (13)
Whole tentacle Extruded
gastrodermal cells
rETRmax
0
10
20
30
40
50
60
70 A. xanthogrammica
A. elegantissima
*P=0.03
n.s.
Fig.2. Maximum relative electron transport rates (rETRmax; means ± s.e.m.)
of E. marina in freshly excised whole tentacles of A. xanthogrammica and
A. elegantissima in comparison to symbionts remaining inside gastrodermal
cells but removed from outer (epidermis + mesoglea) tissue layers of a
second set of freshly excised tentacles.
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2251Host tissues moderate symbiont stress
DISCUSSION
Measurements of E. marina chlorophyll fluorescence and host tissue
properties indicate that symbionts in A. xanthogrammica received
a considerably lower light dose than those in A. elegantissima,
largely because of the attenuation of light by outer tissue layers.
Light attenuation differences are clearly better explained by tissue
thickness than by light-absorbing compounds or pigments. We
suspect that the similar magnitude of differences between the species
in Fv/Fm(35% difference between the means), rETRmax (35%
difference), tissue light attenuation (36% difference) and tissue
thickness (43% difference) is not a coincidence. Collectively, the
data suggest that A. xanthogrammica symbionts received at least
one-third less light than symbionts in A. elegantissima. A higher
degree of light stress related to these differences in light dose is
indicated by the lower Fv/Fmin A. elegantissima, and is analogous
to the effects of light dose on Fv/Fmcommonly observed in reef
corals based on depth and degree of shading (Gorbunov et al., 2001;
Warner et al., 2002; Brown and Dunne, 2008). Reduced Fv/Fmis
symptomatic of the general phenomenon of photoinhibition, a
decline in photochemical efficiency that can be caused by both an
increase in photoprotective processes and damage to PS II (Fitt et
al., 2001; Gorbunov et al., 2001).
In addition to reduced photochemical efficiency, increased light
exposure is typically associated with reduced cellular chlorophyll
content. Higher chlorophyll content in E. marina from A.
elegantissima was unexpected given that they received more light
and had lower photochemical efficiency than symbionts in A.
xanthogrammica. However, slightly larger cell sizes among E.
marina in A. elegantissima may be responsible for their higher
chlorophyll content; chlorophyll fluorescence normalized to relative
cell size showed no host species effect. Cell size in Symbiodinium
and a majority of marine and freshwater phytoplankton has been
found to increase with irradiance (Lesser and Shick, 1989;
Thompson et al., 1991). Another consideration is that E. marina
chlorophyll content does not always change predictably in response
to changes in irradiance. Elliptochloris marina living within A.
elegantissima show little seasonal variation in cell-specific
chlorophyll content despite large fluctuations in irradiance (Verde
and McCloskey, 2007; Dimond et al., 2011). We have also found
that E. marina chlorophyll content within A. elegantissima does not
consistently increase during long-term experimental shading (J.L.D.,
B.L.B. and G. Muller-Parker, unpublished).
A photoprotective function of host tissues has been suggested by
several studies comparing the physiology of algal symbionts in
hospite with that of symbionts removed from the host.
Photosynthesis–irradiance experiments have shown that
Symbiodinium spp. freshly isolated from anemone hosts (Aiptasia
spp.) are more likely to exhibit photoinhibition than those in intact
hosts (Muller-Parker, 1984; Goulet et al., 2005), and that isolated
symbionts are also much more susceptible to heat stress (Goulet et
A. xanthogrammica A. elegantissima
Tissue thickness (μm)
0
100
200
300
400
PAR attenuation
0
0.1
0.2
0.3
0.4
0.5
Epidermis
Mesoglea
Gastrodermis
A
B
C
A. xanthogrammica A. elegantissima
e
m
g
*P=0.002
*P<0.001 Wavelength (nm)
300 400 500 600 700
Absorbance (a.u. mm–3)
0
0.02
0.04
0.06
0.08
0.10
A. elegantissima
A. xanthogrammica
Fig.4. In vitro absorption spectra of host tissue extracts in phosphate buffer
normalized to epidermal tissue volume. Spectra represent upper and lower
bounds of 95% confidence intervals, with confidence intervals of A.
elegantissima superimposed as a dotted line over the A. xanthogrammica
spectrum where they would be covered by spectra overlap. Anthopleura
xanthogrammica tissue extracts absorbed less light per mm3tissue volume
than A. elegantissima extracts, but overlap of 95% confidence intervals
indicates no significant difference between species (P>0.05).
Fig.3. Tissue properties of A. xanthogrammica and A. elegantissima.
(A)Epifluorescence micrographs of representative tentacle sections of the
two host species, showing epidermal green fluorescence from host
fluorescent proteins and gastrodermal red fluorescence from symbiont
chlorophyll. Tissue layers are labeled as follows: e, epidermis; m,
mesoglea; g, gastrodermis. Scale bars, 100m. (B)Thicknesses (means ±
s.e.m.) of epidermis, mesoglea and gastrodermis. (C)Attenuation of
photosynthetically active radiation (PAR; means ± s.e.m.) by strips of
tentacle tissue with gastrodermis removed (epidermis and mesoglea only).
Attenuation is expressed as the proportion of total downwelling PAR
attenuated by host tissues.
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2252
al., 2005). Bhagooli and Hidaka (Bhagooli and Hidaka, 2003)
showed that Fv/Fmof isolated symbionts from several scleractinian
coral species was considerably more sensitive to heat and light stress
than in intact hosts. In those experiments, the key role of light in
symbiont stress physiology was highlighted by the fact that exposure
to elevated temperature in darkness did not affect Fv/Fm, and that
isolated symbionts showed reduced Fv/Fmat lower light levels than
symbionts in intact hosts (Bhagooli and Hidaka, 2003). Thus, intact
host tissues provide critical photoprotection that can moderate the
effects of thermal stress. The photoprotective capacity of host tissues
has been mostly ascribed to fluorescent and non-fluorescent
pigments (Salih et al., 2000; Dove et al., 2008) and UV-absorbing
MAAs (Shick et al., 1995), but here we have demonstrated the
importance of tissue thickness alone.
The attenuation of light by epidermal and mesogleal tissues
accounts for most of the difference in the effective quantum yield
of PS II between the two host species, as shown by measurements
of rETRmax in whole tentacles compared with extruded gastrodermal
cells. The epidermis of both Anthopleura species absorbs and scatters
light via fluorescent and non-fluorescent pigments (Buchsbaum,
1968) and MAAs (Shick et al., 2002). Although the epidermis may
account for much of the photoprotective capacity of outer tissue
layers, we hypothesize that the mesoglea also plays a role through
light scattering. The mesoglea is a matrix of collagen and
mucopolysaccharides, and collagen has a relatively high refractive
index (Johnsen and Widder, 1999). Among several pelagic
cnidarians, Johnsen and Widder (Johnsen and Widder, 1998) found
that mesoglea transparency ranged from 6 to 89% at 400nm.
Notably, of all tissue layers in our study, the mesoglea showed the
greatest difference in thickness (62%) between host species whereas
the epidermis showed the least (29%). In addition to the outer tissue
layers, the gastrodermis of A. xanthogrammica was nearly twice as
thick as that of A. elegantissima, which may also contribute to
photoprotection via the self-shading of successive layers of algal
symbionts in gastrodermal cells.
Host-specific differences in the distribution patterns of
Anthopleura spp. symbionts suggest that the ecological implications
of host tissue thickness are significant. Whereas E. marina is largely
absent from A. elegantissima south of central Oregon (~44°N), in
A. xanthogrammica it occurs approximately 6° farther south into
northern California (~38°N) (Secord and Augustine, 2000).
Similarly, where E. marina co-occurs in the two host species, it is
typically only hosted by A. elegantissima in the low intertidal zone,
whereas in A. xanthogrammica it is hosted much further up the shore
(Secord and Augustine, 2000). Furthermore, E. marina is more
prevalent in A. xanthogrammica even among similar-sized
individuals of the two host species living adjacent to each other in
the same habitat (Bates et al., 2010). The symbiotic dinoflagellate
S. muscatinei, meanwhile, is considerably more prevalent in A.
elegantissima than in A. xanthogrammica. This likely relates to the
higher photophysiological performance of S. muscatinei at high light
and temperature compared with E. marina (Verde and McCloskey,
2001; Verde and McCloskey, 2002). The results of our study
therefore suggest a mechanism for the higher prevalence and
expanded range of E. marina in A. xanthogrammica, particularly
in high-irradiance habitats. Perhaps the distribution and host-
specificity patterns of symbionts on tropical reefs are similarly
influenced by differences in host tissue thickness, as suggested by
LaJeunesse (LaJeunesse, 2002) in reference to differences in host
tissue opacity.
The differences in tissue thickness of the two sea anemone species
undoubtedly relates to their greatly different adult sizes. Anthopleura
elegantissima rarely exceeds a basal diameter of 4cm, whereas A.
xanthogrammica can sometimes exceed 16cm basal diameter
(Sebens, 1981a). This fourfold difference in maximum diameter
equates to a 32-fold difference in biomass based on the equations
of Sebens (Sebens, 1981a) relating diameter to ash-free dry weight.
Using these equations, we estimate that our medium-sized A.
xanthogrammica had a biomass approximately eight times that of
our large-sized A. elegantissima. Back-calculation of tentacle size
scaling from equations of total tentacle surface area and tentacle
number versus ash-free dry weight (Sebens, 1981a) suggest that
scaling in the two species is similar, and that juvenile A.
xanthogrammica the size of adult A. elegantissima have similar-
sized tentacles. Juvenile A. xanthogrammica therefore may not have
thicker tissues than adult A. elegantissima, in which case symbiosis
with E. marina may not be advantageous for juvenile A.
xanthogrammica (or E. marina) living in high-irradiance habitats.
Interestingly, however, juvenile A. xanthogrammica occur almost
exclusively within beds of their principal adult prey, the mussel
Mytilus californianus, remaining in the shaded interstices of these
beds until they reach a basal diameter of approximately 3.5cm
(Sebens, 1981b). This may allow them to initiate and maintain
successful symbioses with E. marina at an early age and small size.
Our study supports the hypothesis that variability in bleaching
susceptibility among reef corals may be related to the
The Journal of Experimental Biology 215 (13)
Table 1. Spectrophotometric and flow cytometric analyses of Elliptochloris marina chlorophyll content in the two host species, Anthopleura
elegantissima and A. xanthogrammica
Host Mean ± s.d. td.f. P
Spectrophotometric analysis
Chl a (pgcell–1)A. elegantissima 1.83±0.21 7.48 9 <0.001
A. xanthogrammica 1.13±0.15
Chl b (pgcell–1)A. elegantissima 0.93±0.13 6.22 9 <0.001
A. xanthogrammica 0.55±0.10
Chl a:chl b A. elegantissima 1.98±0.21 –1.04 9 0.33
A. xanthogrammica 2.08±0.21
Flow cytometric analysis
FL3 (red fluorescence) A. elegantissima 192±28.4 2.89 9 0.02
A. xanthogrammica 166±16.6
FSC (forward scatter) A. elegantissima 1165±92.21 1.93 9 0.09
A. xanthogrammica 1080±127.1
FL3/FSC A. elegantissima 0.166±0.024 0.90 9 0.39
A. xanthogrammica 0.156±0.025
Significant P-values are shown in bold.
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2253Host tissues moderate symbiont stress
photoprotective function of thicker host tissues (Hoegh-Guldberg,
1999; Loya et al., 2001). Bleaching typically involves the synergistic
effects of light and temperature on symbiont photosynthetic
processes (Hoegh-Guldberg, 1999; Weis, 2008), and if one of these
stressors is lessened, the total stress will be reduced. We have shown
that light stress in an algal symbiont can differ by approximately
one-third in relation to host tissue thickness differences of a similar
magnitude. Patterns of bleaching and post-bleaching survival among
different reef coral taxa suggest that variation in tissue thickness
may have profound ecological consequences (Loya et al., 2001; van
Woesik et al., 2011). In Japan, Loya et al. (Loya et al., 2001)
observed that bleaching susceptibility and subsequent mortality after
the 1998 mass coral bleaching event was much lower among
massive, thick-tissued species than in species with thinner tissues
and branching morphologies. Hypotheses for these trends included
enhanced mass transfer efficiency in encrusting or mounding
morphologies versus branching morphologies, and the
photoprotective capacity of thicker tissues (Loya et al., 2001). On
average, tissue thickness of thick- and thin-tissued taxa in their study
differed by 68% (Loya et al., 2001), considerably more than the
differences we measured between Anthopleura species. Although
thin-tissued branching corals provide inter-branch shading and
vertically oriented surfaces that may result in more effective light
attenuation than previously thought (Kaniewska et al., 2011), our
findings show that thicker host tissues alone can provide
considerable symbiont photoprotection. It is likely that variation in
this relatively simple trait has ecological implications ranging from
the biogeography, zonation and host specificity of cnidarian
symbionts to the health and persistence of reef corals in increasingly
warmer seas.
LIST OF ABBREVIATIONS
Fv/Fmmaximum quantum yield of photosystem II
PAM pulse amplitude modulated
PAR photosynthetically active radiation
PS II photosystem II
rETRmax maximum relative electron transport rate
RLC rapid light curve
F/Fm⬘effective quantum yield of photosystem II
ACKNOWLEDGEMENTS
We thank K. Van Alstyne for providing the PAM fluorometer and P. Thut for the
rotary microtome. We are also grateful to G. Muller-Parker and M. Levine for
helpful discussions, and to J. Barber, A. White and three anonymous reviewers for
their critical reading of the manuscript.
FUNDING
This study was supported by the National Science Foundation [IOS-0822179].
B.J.H. was supported by the Centers for Ocean Sciences Education Excellence.
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