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PRIMARY RESEARCH ARTICLE
Elucidating the sponge stress response; lipids and fatty acids
can facilitate survival under future climate scenarios
Holly Bennett
1,2
|
James J. Bell
1
|
Simon K. Davy
1
|
Nicole S. Webster
2,3
|
David S. Francis
4
1
School of Biological Sciences, Victoria
University of Wellington, Wellington, New
Zealand
2
Australian Institute of Marine Science,
Townsville, Queensland, Australia
3
Australian Centre for Ecogenomics, The
University of Queensland, Brisbane,
Queensland, Australia
4
School of Life and Environmental Sciences,
Deakin University, Geelong, Victoria,
Australia
Correspondence
James J. Bell, School of Biological Sciences,
Victoria University of Wellington,
Wellington, New Zealand.
Email: james.bell@vuw.ac.nz
Funding information
Royal Society of New Zealand, Grant/Award
Number: Marsden Fund (VUW1505);
Australian Research Council, Grant/Award
Number: FT120100480; Australian Institute
of Marine Science, Victoria University of
Wellington, the PADI foundation; Deakin
University; VUW Doctoral Scholarship
Abstract
Ocean warming (OW) and ocean acidification (OA) are threatening coral reef
ecosystems, with a bleak future forecast for reef-building corals, which are already
experiencing global declines in abundance. In contrast, many coral reef sponge spe-
cies are able to tolerate climate change conditions projected for 2100. To increase
our understanding of the mechanisms underpinning this tolerance, we explored the
lipid and fatty acid (FA) composition of four sponge species with differing sensitivi-
ties to climate change, experimentally exposed to OW and OA levels predicted for
2100, under two CO
2
Representative Concentration Pathways. Sponges with
greater concentrations of storage lipid, phospholipids, sterols and elevated concen-
trations of n-3 and n-6 long-chain polyunsaturated FA (LC PUFA), were more resis-
tant to OW. Such biochemical constituents likely contribute to the ability of these
sponges to maintain membrane function and cell homeostasis in the face of envi-
ronmental change. Our results suggest that n-3 and n-6 LC PUFA are important
components of the sponge stress response potentially via chain elongation and the
eicosanoid stress-signalling pathways. The capacity for sponges to compositionally
alter their membrane lipids in response to stress was also explored using a number
of specific homeoviscous adaptation (HVA) indicators. This revealed a potential
mechanism via which additional CO
2
could facilitate the resistance of phototrophic
sponges to thermal stress through an increased synthesis of membrane-stabilizing
sterols. Finally, OW induced an increase in FA unsaturation in phototrophic sponges
but a decrease in heterotrophic species, providing support for a difference in the
thermal response pathway between the sponge host and the associated photosym-
bionts. Here we have shown that sponge lipids and FA are likely to be an important
component of the sponge stress response and may play a role in facilitating sponge
survival under future climate conditions.
KEYWORDS
climate change, coral reef, fatty acid, heterotroph, lipid, mechanism, ocean acidification, ocean
warming, phototroph, porifera
Received: 28 November 2017
|
Revised: 10 February 2018
|
Accepted: 12 February 2018
DOI: 10.1111/gcb.14116
Glob Change Biol. 2018;1–15. wileyonlinelibrary.com/journal/gcb ©2018 John Wiley & Sons Ltd
|
1
1
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INTRODUCTION
As the climate changes, ocean warming (OW) and ocean acidification
(OA) pose a number of threats to coral reefs (Heron, Maynard, &
Ruben Van Hooidonk, 2016; Hughes et al., 2017; Manzello, Eakin, &
Glynn, 2017). Climate change conditions projected for 2,100 com-
bined with ongoing degradation from local stressors, are expected to
cause significant declines in coral cover and create space for other
more tolerant organisms (Bell, Davy, Jones, Taylor, & Webster, 2013;
Kroeker, Micheli, & Gambi, 2013; Norström, Nyström, Lokrantz, &
Folke, 2009). Some coral reef sponges are able to tolerate elevated
temperature and oceanic pCO
2
, suggesting a capacity to proliferate
on coral reefs as space is made available by declines in more sensitive
reef species (Bell et al., 2013; Bennett et al., 2017; Duckworth &
Peterson, 2013; Duckworth, West, Vansach, Stubler, & Hardt, 2012;
Fang et al., 2013; Lesser, Fiore, Slattery, & Zaneveld, 2016; Stubler,
Furman, & Peterson, 2015; Vicente, Silbiger, Beckley, Raczkowski, &
Hill, 2015; Wisshak, Sch€
onberg, Form, & Freiwald, 2013). To date, cli-
mate change research on marine sponges has focused primarily on
the physiological response of these sessile organisms to predicted
OW and OA. However, while physiological responses measure the
systemic tolerance of sponges and reflect their ability to acclimate to
environmental change, the mechanisms underpinning these responses
remain unclear. Mechanistic understanding is required to assess the
acclimatization and adaptation potential of this important phylum in
the face of environmental change (Putnam, Barott, Ainsworth, &
Gates, 2017). Furthermore, OW and OA are known to influence other
cellular and molecular processes that may not be reflected by host
physiology during experimental exposure (P€
ortner, 2008), and it is
likely that adjustments at the molecular or membrane level will ulti-
mately define tolerance limits (P€
ortner, 2002).
The lipid bilayer (cell membrane), which forms a permeable barrier
for cells and subcellular organelles, is sensitive to environmental stres-
sors (Hazel, 1995). This is particularly apparent in relation to tempera-
ture, where changes will alter cell membrane fluidity and cytotoxicity
(Parrish, 2013; V
ıgh et al., 2007). At an organism’s adapted tempera-
ture, lipids in this bilayer (membrane lipids) are in the liquid-crystalline
phase, and enable vital cellular functions including the regulation of
transmembrane activities (e.g. nutrient transport); the maintenance of
solute gradients for energetic processes; and the facilitation of enzyme
activity by providing a matrix where biochemical reactions can occur
(Guillot, Obis, & Mistou, 2000; Hazel, 1995; Neidleman, 1987; Sinen-
sky, 1974). As temperature increases, the movement of fatty acid (FA)
acyl chains increases and membrane lipids assume a disordered
inverted hexagonal phase. As such, beyond an organism’s optimal tem-
perature range, membranes become “hyperfluid”, resulting in a loss of
bilayer integrity, which subsequently compromises cell homeostasis
and overall cell function (Hazel, 1995).
Ectothermic organisms, including sponges, can counteract the
effects of increased temperature through the compositional alter-
ation of membrane lipids in a process called homeoviscous adapta-
tion (HVA) (Martin-Creuzburg & Elert, 2009; Nes, 1974; Volkman,
2003). HVA involves changes in the mechanical and chemical proper-
ties of the lipid bilayer to produce membranes with constant fluidity
as temperature changes (Horv
ath et al., 2012; Parrish, 2013;
Sajbidor, 1997; Sinensky, 1974; V
ıgh et al., 2007; Weirich & Reigh,
2001). Under thermal stress, organism-specific responses are
employed to prevent membrane destabilization and to maintain the
“ideal”functional state of the cell membrane (Guerzoni, Lanciotti, &
Cocconcelli, 2001). Such adaptive mechanisms may involve: (i) shifts
in the relative proportion of membrane lipids (phospholipids, glycol-
ipids and sterols), where sterols are particularly important for main-
taining membrane rigidity at super-optimal temperatures (Copeman
& Parrish, 2004; Parrish, 2013); (ii) increasing FA chain length to
reduce the fluidity of cell membranes (Guerzoni et al., 2001;
Hochachka & Somero, 1984); (iii) increasing the proportion of satu-
rated FA (SFA) in relation to polyunsaturated FA (PUFA) with tem-
perature, as SFA are more resistant to lipid oxidation and facilitate
greater membrane stability (Tchernov et al., 2004; Wada, Gombos, &
Murata, 1994); and (iv) reducing the degree of FA saturation and
increasing membrane fluidity, whereby unsaturated FA are less likely
to pack together in the bilayer given their “kinked”structure (Hazel,
1995).
Sponge lipid composition is distinctive among marine organisms
(Lawson, Stoilov, Thompson, & Djerassi, 1988), particularly in relation
to their lipid bilayer, which contains an abundance of novel phospho-
lipids and sterols, and a high diversity of FA. Sponges have an
abundance of long-chain C
22
-C
30
FA with the presence of branched,
odd-chain, or hydroxyl groups (Koopmans et al., 2015), commonly
referred to as the “demospongic acids”despite more recent evidence
confirming their biosynthesis is not specific to sponges (Berg
e & Bar-
nathan, 2005; Djerassi & Lam, 1991; Koopmans et al., 2015; Korn-
probst & Barnathan, 2010; Rod’kina, 2005). Such long-chain FA are
particularly important for environmental stress resistance, due to the
role that they play in maintaining the fluidity of cell membranes and
controlling cellular responses to external stimuli, including changing
temperatures (Arts & Kohler, 2009; Berg
e & Barnathan, 2005; Hix-
son & Arts, 2016; Parrish, 2013). These features of sponge lipid and
FA composition may play at least some part in the persistence of
sponges in unpredictable and variable environments (Djerassi & Lam,
1991; Genin et al., 2008; Lawson et al., 1988; Santalova et al.,
2004). However, while it is understood that sponges can control the
fluidity of their cell membranes with changing temperatures season-
ally, e.g. by increasing the concentration of lipids that have a higher
melting point in summer (Lawson et al., 1988), there is a lack of
studies considering the effects of environmental change on sponge
lipid and FA content (Arillo, Bavestrello, Burlando, & Sara, 1993).
Previous work in which four abundant Great Barrier Reef (GBR)
sponge species—the phototrophic Carteriospongia foliascens and
Cymbastela coralliophila and the heterotrophic Rhopaloeides odorabile
and Stylissa flabelliformis—were experimentally exposed to OW and
OA, revealed species-specific differences in tolerance to these stres-
sors (Bennett et al., 2017), although the mechanisms underpinning
the responses were not resolved. Elevated pCO
2
was also found to
2
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BENNETT ET AL.
provide the phototrophic sponges with protection from thermal
stress; however, the pathways underlying this ameliorative effect
also remained unclear (Bennett et al., 2017). Therefore, although we
are beginning to understand sensitivity thresholds of sponges
exposed to OW and OA, little is known about the mechanisms that
enable them to cope with such environmental impacts. The physical
properties of lipids within cell membranes, and the ability of organ-
isms to regulate and adapt their cell lipid bilayers in response to
thermal stress play a significant role in stress tolerance, and ulti-
mately define an organism’s thermal limits (Arts & Kohler, 2009; Gei-
der, 1987; Hazel, 1995; Singh, Sinha, & Hader, 2002). Here, we
assessed the lipid and FA composition of the phototrophic sponges
Carteriospongia foliascens and Cymbastela coralliophila, and the het-
erotrophic sponges Rhopaloeides odorabile and Stylissa flabelliformis,
before and after experimental exposure to OW and OA, and
explored HVA mechanisms to ascertain whether more tolerant spe-
cies are able to alter membranes to acclimate to OW through
changes in lipid and FA composition.
2
|
MATERIALS AND METHODS
2.1
|
Experimental design
The experimental design is described in detail in Bennett et al.
(2017). Briefly, sponges were exposed to nine combined temperature
and pH treatments. Treatments were based on present day CO
2 atm
levels (~400 ppm) and projected CO
2 atm
increases for 2,100 under
the IPCC “baseline”emission scenarios RCP6.0 (~800 ppm) and
RCP8.5 (~1,200 ppm) (IPCC, 2014). The experiment was performed
within the National Sea Simulator at the Australian Institute of Mar-
ine Science (AIMS). Target treatments for the experiment were 28.5,
30 and 31.5°C and pH (Total scale) 8.1, 7.8 and 7.6. All factors were
fully crossed, resulting in nine experimental treatments and three
replicates of each treatment.
Sponges collected from 10 to 15 m depth at Davies Reef on the
Great Barrier Reef, Australia (18°820S, 147°650E). Due to the large
size of adult sponges, for all species except C. foliascens,~20 larger
specimens were cut to form ~60 smaller clones. For C. foliascens,
~30 small individuals and 15 large individuals (each cut into 2–3
clones) were collected. Once healed, clones were treated as “individ-
ual”sponges and were randomly allocated to experimental tanks
(maintained at 27°C and pH 8.1 =T0/ambient). A 12-week exposure
period post-ramping was planned for all adult sponges, with experi-
ments terminated on a species-by-species basis as lethal effects
were observed (see Bennett et al., 2017 for more details).
2.2
|
Tissue sampling
Six sponges of each species were sacrificed at time zero (T0) for ini-
tial tissue analysis. Tissue was then taken from experimental sponges
at the final sampling point (n=4 to 6 individuals per species per
treatment, except where mortalities occurred (C. foliascens where
n=2 at 31.5°C/pH 8.1 and 31.5°C/pH 7.8; and R. odorabile where
n=2 at 31.5°C/pH 7.6). The final sampling point varied for each
species, due to differing sensitivities to the RCP8.5 treatment condi-
tions. The experiment was terminated after T=2 weeks for the
“sensitive”species, C. foliascens and R. odorabile, which were sensi-
tive to RCP8.5 OW (C. foliascens) and the combined effects of OW
and OA (R. odorabile). The more “tolerant”species, S. flabelliformis
and C. coralliophila, resisted RCP8.5 conditions for significantly
longer. The experiment was terminated after T=8 weeks for S. fla-
belliformis following high levels of tissue necrosis at 31.5°C and
C. coralliophila remained in the experiment for the full 12 week
exposure, despite high levels of bleaching at 31.5°C. Sponge tissue
was cryopreserved in liquid nitrogen in 1.5 ml vials for subsequent
chlorophyll a, total lipid, lipid class and FA analysis.
2.3
|
Chlorophyll adetermination (phototrophic
species only)
Chlorophyll a(Chl a) concentrations were determined for the two
phototrophic species as a proxy for the presence of phototrophic
symbionts, and therefore an index of sponge bleaching (Wilkinson,
1983), following the methods of Pineda, Duckworth, and Webster
(2016). Chl awas extracted from approximately 50 mg of cryopre-
served sponge tissue in 95% ethanol with a total of 1.4 ml pigment
extract recovered from each sample. Triplicate 300 ll extracts of
each sample were analysed on a Power Wave Microplate Scanning
Spectrophotometer (BIO-TEKw Instruments Inc., Vermont, USA). Chl
aconcentrations were normalized to sponge wet weight as: Chl a
(mg/ml) 9extraction volume (ml)/wet weight (g).
2.4
|
Total lipid
Lipids were extracted from 20 to 600 mg of freeze-dried and
crushed sponge tissue, according to the method described by Folch,
Less, and Sloane-Stanley (1957), following modifications by Conlan,
Jones, Turchini, Hall, and Francis (2014). Samples were sonicated
(Vibracell, Sonics and Materials, Newtown, USA), and then filtered
into a scintillation vial. This process was repeated three times, result-
ing in ~9 ml of filtrate, to which 4.5 ml of a potassium chloride sam-
ple wash [KCl (0.44%) in H
2
O (3)/CH
3
OH (1)] was added. The
mixture was incubated for 18 hr at room temperature, after which
the bottom layer containing the extracted lipid was recovered and
the solvent was evaporated under nitrogen. Total lipid content was
weighed and standardized to dry weight and expressed in mg lipid
per g freeze-dried sponge dry weight. Once lipid content was deter-
mined, the lipid fraction was resuspended in 1 ml dichloromethane
for subsequent lipid class analysis.
2.5
|
Lipid class analysis
Lipid class analysis followed the method described by Nichols,
Mooney, and Elliot (2001), with modifications by Conlan et al.
(2014). A 100 ll aliquot of the resuspended total lipid fraction was
taken and analysed for lipid class composition using thin layer
BENNETT ET AL.
|
3
chromatography and flame ionization detection (Iatroscan MK 6s,
Mitsubishi Chemical Medience Tokyo Japan). Samples were spotted
in duplicate onto silica gel S4-chromarods (5 lm particle size). Lipid
separation followed a two-step sequence: (i) the elution of the phos-
pholipids (PL), phosphatidylethanolamine (PE), phosphatidylserine-
phosphatidylinositol (PS-PI) and phosphatidylcholine (PC) in a
dichloromethane/methanol/water (50:20:2, by volume) solvent sys-
tem; and (ii) after air drying, the elution of the acetone mobile polar
lipids (AMPL), sterols (ST), sterol esters (WE), triacylglycerols (TAG),
free fatty acids (FFA), and 1,3-diacylglycerol (DG) in a hexane/diethyl
ether/formic acid (60:15:1.5, by volume) solvent system. The Iatros-
can MK 6s was calibrated using known compound classes in the
range of 0.1–10 lg (Sigma-Aldrich, Inc., St. Louis, MO, USA and from
Nu-Chek Prep Inc., Elysian, MN, USA) and peaks were quantified
using POWERCHROM version 2.6.15 (eDAQ Pty Ltd.). The contribution
of each lipid class was standardized to mg lipid class per g lipid. Lipid
classes were grouped as ‘structural’and ‘storage’, depending on their
primary functional roles. PL (PC, PS-PI, PE), AMPL and ST were com-
bined as the structural lipid component, and WE, TAG, FFA and DG
were combined as the storage lipids. The effects of OW and OA on
individual lipid classes were also explored.
2.6
|
Fatty acid analysis
FA were esterified into methyl esters using the acid-catalysed
methylation method (Christie, S
eb
edio, & Juan
eda, 2001) with modi-
fications described by Conlan et al. (2014). 100 ll of internal stan-
dard (0.378 mg/ml, C23:0; Sigma-Aldrich, Inc., St. Louis, MO, USA)
were added to a 100 ll aliquot of the total lipid fraction with 2.0 ml
of the methylation catalyst, acetyl chloride: methanol (1:10). The
resultant hexane supernatant, containing the FA extraction, was
recovered into a gas chromatography (GC) vial for GC injection.
Fatty acid methyl esters were isolated and identified using an Agilent
Technologies 7890B GC System (Agilent Technologies, USA)
equipped with a BPX70 capillary column (120 m 90.25 mm internal
diameter, 0.25 mm film thickness, SGE Analytical Science, Australia),
a flame ionization detector (FID), an Agilent Technologies 7693 auto
sampler, and a splitless injection system. The injection volume, injec-
tor and detector temperatures, and temperature programmes fol-
lowed Conlan et al. (2014); the carrier gas was helium at 1.5 ml/min
at a constant flow. Individual FA were then identified using known
external standards (a series of mixed and individual standards from
Sigma-Aldrich, Inc., St Louis, USA and Nu-Chek Prep Inc., USA), using
the software GC CHEMSTATION (Rev B.04.03, Agilent Technologies). The
resulting peaks were corrected by theoretical relative FID response
factors (Ackman, 2002) and quantified relative to the internal stan-
dard C23:0.
FA content was standardized to weight of total lipid content for
each sample and expressed as mg FA per g lipid. Total FA content
and sums of major FA classes: saturated FA (SFA); monounsaturated
FA (MUFA); polyunsaturated FA (PUFA); trans-unsaturated FA
(TRANS-FA); omega-3 PUFA (n-3 PUFA); omega-6 PUFA (n-6 PUFA);
omega-3 long-chain PUFA (n-3 LC PUFA) and omega-6 long-chain
PUFA (n-6 LC PUFA) were calculated to explore how sponge FA
content varied between species and how the FA profiles of sponges
was affected by OW and OA.
2.7
|
Homeoviscous adaptation mechanisms
The following calculations were also made to explore potential HVA
mechanisms in response to the treatments:
(1) The ratio of sterol to phospholipid (sterol:phospholipid)
(2) The ratio of structural to storage lipid (structural:storage)
(3) Mean chain length (MCL) following Guerzoni et al. (2001):
MCL ¼Rðmg FA g lipid1CÞ=total mg FA g lipid1
(4) The ratio of SFA to PUFA (SFA:PUFA)
(5) The degree of FA unsaturation (DoU) following Guerzoni et al.
(2001):
DoU ¼½Rmonoenes þ2ðRdienesÞþ3ðRtrienesÞ
þ4ðRtetraenesÞþ5ðRpentaeneÞ
þ6ðRhexaenesÞ=total FA
2.8
|
Data analysis
Data analyses were performed with PRIMER-E(PRIMER version 6.0, PER-
MANOVA+, Plymouth Marine Laboratory, Plymouth, UK). All graphs
were generated using GRAPHPAD PRISM (GRAPHPAD Software, version
6.00 for Windows, La Jolla California USA). For all analyses, Eucli-
dean distances were used to generate a resemblance matrix. All mul-
tivariate data were standardized prior to generating the resemblance
matrix. Permutational post hoc comparisons were used to determine
which treatments (species, temperature, pH) differed significantly. A
5% significance level was used for all tests.
2.8.1
|
Univariate analysis
To test the effect of temperature and pH on chlorophyll aconcen-
tration and HVA mechanisms, a two-way Permutational Multivariate
Analysis of Variance (PERMANOVA) was employed, with tempera-
ture and pH as fixed factors.
2.8.2
|
Multivariate analysis
To determine whether lipid and FA composition were significantly
different between species at time zero, a one-way PERMANOVA
was conducted on a matrix of total lipid and standardized lipid class
data, total FA, FA classes and individual FA data, with species as a
fixed factor. To test and visualize the effect of temperature and pH
on sponge lipid and FA profiles a two-way PERMANOVA was con-
ducted on a matrix of total lipid and standardized lipid class data
4
|
BENNETT ET AL.
(lipid profiles) and a matrix of total FA, FA classes and individual FA
data (FA profiles) for each species following exposure to the treat-
ments, with temperature and pH as fixed factors. CAP was used to
display significant differences in lipid and FA profiles between
species and treatments. SIMPER analysis identified the individual FA
contributing to differences in sponge FA profiles between
treatments.
3
|
RESULTS
3.1
|
The lipid and FA composition of four sponge
holobionts
PERMANOVA revealed significant differentiation in the lipid and
FA composition between the four sponge species (Pseudo-F(3,
20) =14.05, p=.001), which was also clearly evident in the CAP
ordination (Figure 1). The profiles of the phototrophic sponges
was similar (p=.066), both species had low total lipid content (50
to 54 mg lipid g sponge
1
for C. foliascens and C. coralliophila,
respectively). In contrast, the heterotrophic sponges had a higher
concentration of total lipid (141 and 159 mg lipid g sponge
1
for
R. odorabile and S. flabelliformis respectively). Interestingly, the
profiles of the two sensitive species, C. foliascens and R. odorabile,
were similar (p=.073) due to a significant contribution of AMPL
to their total lipid concentration (Figures 1 and 2a). Furthermore,
although the overall profiles of the two more tolerant species,
C. coralliophila and S. flabelliformis, were significantly different
(p=.003), their profiles were both characterized by higher con-
centrations of storage lipids, phospholipids and sterols (Figures 1
and 2a).
The two phototrophic sponges and S. flabelliformis had similar
concentrations of total FA (70, 68 and 71 mg g lipid
1
for C. folias-
cens,C. coralliophila, and S. flabelliformis respectively), almost double
that of the heterotrophic R. odorabile (45 mg g lipid
1
). Both pho-
totrophic sponges had an abundance of SFA (16:0 in particular), as
well as a high concentration of the MUFA 16:1n-7 and 18:1n-9 (Fig-
ures 1 and 2b; Table S1), whereas the heterotrophic sponge FA pro-
files were significantly different from each other (p=.003). The
profile of R. odorabile was distinguished by greater concentrations of
trans FA (Figures 1 and 2b), whereas S. flabelliformis was character-
ized by a high concentration (6.5 mg g lipid
1
) of arachidonic acid
(ARA; 20:4n-6) and 26:2n-17 (32 mg g lipid
1
; Table S1). The FA
profiles of the two more tolerant species, C. coralliophila and S. fla-
belliformis (S. flabelliformis in particular) were characterized by higher
bioactive n-3 and n-6 LC PUFA (Figure 1). C. coralliophila had the
lowest PUFA concentration (Figure 2b), and proportionately the
highest contribution of bioactive n-3 and n-6 PUFA (Figure 2c).
3.2
|
Chl a—bleaching index
Chl aconcentrations in the two phototrophic sponges declined with
increasing temperature (Figure 3a-b). C. foliascens Chl aconcentra-
tions were significantly lower at 31.5°C compared to 28.5°C
(p=.011), and C. coralliophila Chl aconcentrations declined signifi-
cantly at both 30°C(p=.004) and 31.5°C(p<.001).
3.3
|
Sponge lipid and FA profiles under OW and
OA
There was no significant effect of pH or temperature on the lipid
class profiles of the four sponge species (Table 1). However, temper-
ature had a significant effect on the FA profile of all species, except
S. flabelliformis (Table 1). The FA profile of bleached C. foliascens (as
assessed by reduced Chl aconcentrations above) exposed to 31.5°C
differed significantly from that of sponges in the lower temperature
treatments (28.5°C: p=.015; 30°C: p=.029). Sponges at 31.5°C
had a lower total FA content (32.5 mg g lipid
1
) than sponges at
28.5°C (33.1 mg g lipid
1
) and 30°C (33.4 mg g lipid
1
), primarily
due to a reduction in SFA and MUFA, including SFA 16:0; MUFA
16:1 n-7; and the demospongic acids 5,12-Me 18:2n 9, 5,9-Me
FIGURE 1 Canonical analysis of principal coordinates (CAP)
ordination showing the lipid and FA composition of four GBR
sponge species prior to exposure to experimental treatments. One-
way PERMANOVA and post hoc pairwise comparisons identified
significant differences in lipid and FA composition between the four
species. Each point on the CAP represents an individual sponge
where =Carteriospongia foliascens;=Cymbastela coralliophila;
=Rhopaloeides odorabile; and =Stylissa flabelliformis. The lipid
and FA classes correlated with the differences between groups are
overlaid. WE =wax ester, TAG =triacylglycerol, FFA free fatty
acids, ST =sterol, AMPL =acetone mobile polar lipid,
PL =phospholipid (PS +PC +PI +PE), SFA =saturated fatty acid;
MUFA =monounsaturated fatty acid; PUFA =polyunsaturated fatty
acid; and TRANS =trans fatty acid, LC =long chain
BENNETT ET AL.
|
5
FIGURE 2 Time zero (T0) lipid and FA class composition of each species presented as (a) Average (+SE) mg lipid per gram of sponge
(mg lipid sponge
1
). Lipid classes are coded as follows: (i) storage lipid: WE =wax ester, TAG =triacylglycerol, FFA =free fatty acids, (note
1,3-diacylglycerol (DG) was detectable in trace amounts in C. coralliophila only at T0 and is not presented here); and (ii) structural lipid:
ST =sterol, AMPL =acetone mobile polar lipid, PL =phospholipid (PS +PC +PI +PE). (b) Average (+SE)FAper mg of lipid (FA mg g lipid
1
).
Major FA classes are coded as follows: SFA =saturated fatty acid; MUFA =monounsaturated fatty acid; PUFA =polyunsaturated fatty acid;
and TRANS =trans fatty acid). (c) PUFA composition, presented as the relative proportion (%) of each PUFA type (n-6, n-3 n-6 long chain and
n-3 long chain, and other PUFA) to the total PUFA content for each species (n=6 to 9 sponges per species). P =phototrophic species and
H=heterotrophic species
FIGURE 3 Chlorophyll aconcentration for (a) Carteriospongia foliascens and (b) Cymbastela coralliophila. Values are mean lg chlorophyll a
per gram of sponge wet weight (n=4 to 6 for C. foliascens, and n=6 for C. coralliophila) and mean P:R ratio (n=6per treatment—except
where mortalities occurred) SE for each treatment. P =phototrophic species
6
|
BENNETT ET AL.
18:2n 9 and 5,9-Me 24:5 n-3 (Figure 4a). The FA profile of sponges
at 31.5°C was further distinguished from those in the lower tem-
perature treatments by a higher concentration of the SFA 6:0 and
22:0,aswellasthen-6 PUFA 18:3 n-6, and n-3 LC PUFA including
22:5 n-3 (DPA; Figure 4a). It is interesting to note here that,
although the concentration of DPA increased in sponges at 31.5°C,
the concentration of other FA, including 20:4n-6 (ARA) and 20:5
n-3 (EPA), declined by almost half relative to the concentration at
28.5°C.
The FA profile of C. coralliophila at 28.5°C differed from the FA
profile of bleached sponges at 31.5°C(p=.001). As with C. folias-
cens, this difference was characterized by a reduction in SFA and
MUFA content in bleached sponges, driven primarily by declines in
SFA 16:0 and the MUFA 14:1 n-5, 16:1 n-7 and 18:1 n-9 (Fig-
ure 4b), although 17-Me 26:1 n-9 increased in these sponges. Mean-
while, the concentration of SFA 6:0 and 14:0 increased in sponges
at 31.5°C (Figure 4b). Similar to bleached C. foliascens, the PUFA
content of C. coralliophila increased in sponges exposed to 31.5°C,
with 22:2 n-6 and 22:4 n-6 contributing significantly to this differ-
ence (Figure 4b). Interestingly, the FA profile of C. coralliophila at
28.5°C also differed from sponges exposed to 30°C(p=.004) as a
result of an increase in SFA 3, 7, 11, 15-tetra-Me 16:0 and a higher
PUFA content in these sponges, with the n-6 LC PUFA 22:2 n-6 and
22:5 n-6 making a significant contribution to this increase
(Figure 4b).
The FA profile of R. odorabile at 28.5°C differed significantly
from that of sponges at 31.5°C(p=.019) due to an increase in
MUFA, as despite a number of MUFA declining at 31.5°C (14:1 n-5,
17:1 n-7 and 22:1 isomers), 17-Me 26:1 n-9 increased by more than
50% in thermally stressed sponges (Figure 4c). Further to this, trans
FA 18:1 n-9t declined at 31.5°C, as did three PUFA: 16:3 n-4; 23-
Me 5, 9 24:2 n-17; and 26:2 n-17 (Figure 4c). Meanwhile, and similar
to the thermally sensitive C. foliascens, the concentration of 20:4 n-6
(ARA) declined by almost half compared to that in sponges at
28.5°C. There was no significant treatment effect on the FA profile
of S. flabelliformis.
3.4
|
Sponge homeoviscous adaptation mechanisms
There was no significant effect of OW or OA on the ratio of struc-
tural to storage lipids across all species (Table 2; Figure 5b,c). There
was, however, a significant pH effect on the ratio of ST: PL in C. fo-
liascens (Table 2; Figure 5a). This ratio increased significantly when
sponges were exposed to reduced pH (Figure 5a). There was a sig-
nificant effect of temperature on mean chain length (MCL) for the
heterotrophic sponge R. odorabile (Table 2; Figure 5c). MCL of FA
increased with exposure to increased temperature, with sponges at
31.5°C having significantly higher MCLs than those at 28.5°C. The
degree of FA unsaturation (DoU) increased significantly with temper-
ature for both phototrophic species, C. foliascens and C. coralliophila
(Table 2; Figure 5d). In contrast, DoU decreased significantly for the
heterotrophic species R. odorabile with OW, and although nonsignifi-
cant, S. flabelliformis DoU followed a downward trend at 31.5°C/pH
7.6 (Table 2; Figure 5d). The ratio of SFA to PUFA subsequently
decreased significantly with temperature for C. coralliophila (Table 2;
Figure 5d) and was lowest under ambient pH in the highest temper-
ature treatment for C. foliascens (Table 2; Figure 5d), however, this
was not significant.
TABLE 1 Results of 2-factor PERMANOVA testing the effects of pH and temperature of sponge lipid and FA profiles
C. foliascens R. odorabile S. flabelliformis C. coralliophila
df F p df F p df F p df F p
Lipid
Temp 2, 27 2.38 .118 2, 42 0.35 .908 2, 27 0.56 .703 2, 38 0.78 .607
pH 2, 27 2.35 .095 2, 42 0.62 .680 2, 27 0.75 .524 2, 38 0.71 .675
Temp*pH 4, 27 2.44 .067 4, 42 0.86 .545 4, 27 0.40 .944 4, 38 0.68 .812
Fatty acid
Temp 2, 27 2.90 .012 2, 42 3.28 .047 2. 27 0.86 .519 2, 38 5.14 .001
pH 2, 27 1.46 .180 2, 42 0.86 .431 2. 27 1.24 .317 2, 38 1.15 .322
Temp*pH 4, 27 1.79 .060 4, 42 0.22 .973 4. 27 0.62 .849 2, 38 0.95 .516
Significant p-values (p<.5) are bolded.
FIGURE 4 Canonical analysis of principal coordinates (CAP) ordination (left) of sponge FA profiles following exposure to OW and OA, and
bar graph (right) showing main FA contributing to differences in sponge FA profiles between treatments. Two-way PERMANOVA and post hoc
pairwise comparisons identified significant differences in FA composition for (a) Carteriospongia foliascens (b) Cymbastela coralliophila and (c)
Rhopaloeides odorabile. Each point on the CAP represents an individual sponge exposed to a different OW/OA treatment where =28.5°C/
pH 8.1, =28.5°C/pH 7.8, =28.5°C/pH 7.6, =30°C/pH 8.1, =30°C/pH 7.8, =30°C/pH 7.6, =31.5°C/pH 8.1, =31.5°C/pH
7.8, =31.5°C/pH 8.6 (n=3 to 6 individuals per species per treatment, except where mortalities occurred for R. odorabile where n=2at
31.5°C/pH 7.6). As there was no significant treatment effect on the S. flabelliformis FA profile, a CAP ordination is not presented
BENNETT ET AL.
|
7
8
|
BENNETT ET AL.
4
|
DISCUSSION
Exploration of sponge lipid and FA composition revealed indicators
of intrinsic sponge tolerance to OW and OA, provided insight into
the types of sponges that will survive in a warmer, high CO
2
ocean,
and identified potential mechanisms of climate change acclimation in
sponges. While it is well established that lipids and FA play an
important role in stress resistance, and the ability of an organism to
maintain appropriate membrane function in the face of environmen-
tal change is intimately linked to tolerance, this is the first time that
these responses have been demonstrated in sponges.
4.1
|
Innate sponge tolerance
The two phototrophic sponges had similar lipid and FA profiles due
to an abundance of FFA, triacylglycerides and SFA. While this is
likely reflective of their specific mode of nutrition, future work
exploring the FA profiles of the sponge host and associated sym-
bionts in isolation will be needed to confirm this (Berg
e & Barnathan,
2005; Wada & Murata, 1998). What is of particular interest here,
however, are the different lipid and FA profiles that separate the
sensitive species, C. foliascens and R. odorabile, from the more toler-
ant species, C. coralliophila and S. flabelliformis. The thermally resis-
tant sponges had a high storage lipid content, due to a greater
contribution of WE. This storage lipid may facilitate resistance of
these species to OW and OA by providing energy during periods of
stress (Anthony, Hoogenboom, Maynard, Grottoli, & Middlebrook,
2009; Kattner & Hagen, 2009). Wax esters may also function as
structural elements, providing cell membrane support and possibly
serving as FA carriers in the biosynthesis of structural lipids (Mars-
den, 1975; Nevenzel, 1970; Parrish, 1988); such features likely also
assist resistance to environmental stress. Meanwhile, the more sensi-
tive species had a high concentration of AMPL. This group of lipids
contains pigments, glycolipids and monoacylglycerols (Murata & Sie-
genthaler, 2006; Parrish, 2013), which are found in abundance in
bacterial lipids (Shaw, 1974), reflecting the higher microbial content
of these sponge species (Luter et al., 2015; Moitinho-Silva et al.,
2017). Glycolipids in particular are important for membrane stability
(H€
olzl & D€
ormann, 2007) and may play a role in facilitating bacterial
survival within the sponge host, where environmental conditions can
be variable (Thomas et al., 2010).
The more thermally resistant species also had a higher concen-
tration of sterols and phospholipids, the primary constituents of the
lipid bilayer. These structural lipids are fundamental for cell support
and protection, and help to maintain membrane fluidity under stress-
ful conditions (Lawson et al., 1988; Murata & Siegenthaler, 2006;
Paulucci, Medeot, Dardanelli, & De Lema, 2011); they also likely pro-
vide a key indicator of environmental stress tolerance in these
sponges (Tchernov et al., 2004). Furthermore, the more thermally
resistant heterotroph, S. flabelliformis, had a high concentration of
TABLE 2 Results of 2-factor PERMANOVA testing the effect of pH and temperature on sponge HVA mechanisms
C. foliascens R. odorabile S. flabelliformis C. coralliophila
df F p df F p df F p df F p
ST: PL
Temp 2, 27 0.60 .596 2, 42 1.14 .30 2, 27 1.19 .305 2, 38 0.43 .636
pH 2, 27 5.01 .012 2, 42 1.17 .31 2, 27 0.42 .672 2, 38 0.98 .396
Temp*pH 4, 27 1.20 .341 4, 42 0.77 .59 4, 27 0.55 .716 4, 38 1.10 .355
Struc: Store
Temp 2, 27 0.36 .696 2, 42 0.11 .89 2, 27 0.70 .493 2, 38 1.45 .255
pH 2, 27 2.55 .084 2, 42 0.12 .88 2, 27 0.34 .724 2, 38 <0.01 .927
Temp*pH 4, 27 1.72 .173 4, 42 0.45 .77 4, 27 0.18 .955 4, 38 0.16 .959
MCL
Temp 2, 27 0.51 .599 2, 42 4.32 .019 2, 27 1.22 .322 2, 38 1.56 .210
pH 2, 27 1.12 .343 2, 42 0.44 .622 2, 27 1.75 .162 2, 38 0.69 .485
Temp*pH 4, 27 1.03 .405 4, 42 0.22 .937 4, 27 0.53 .826 4, 38 1.06 .398
DoU
Temp 2, 27 3.29 .046 2, 42 3.53 .035 2, 27 0.80 .492 2, 38 4.35 .022
pH 2, 27 0.38 .679 2, 42 0.88 .443 2, 27 0.16 .890 2, 38 2.16 .117
Temp*pH 4, 27 2.08 .112 4, 42 0.19 .940 4, 27 0.68 .696 4, 38 2.43 .054
SFA: PUFA
Temp 2, 27 0.72 .519 2, 42 3.01 .059 2. 27 1.01 .443 2, 38 4.52 .023
pH 2, 27 1.20 .306 2, 42 0.07 .924 2. 27 1.05 .430 2, 38 0.49 .490
Temp*pH 4, 27 2.82 .052 4, 42 0.33 .851 4. 27 0.73 .725 2, 38 1.35 .268
Significant p-values (p<.5) are bolded.
BENNETT ET AL.
|
9
n-3 and n-6 LC PUFA. Similarly, while the more thermally resistant
phototrophic C. coralliophila had a low PUFA content, over half of
these PUFA were bioactive n-3 and n-6 LC PUFA. This class includes
biologically important FA, which are significant constituents of phos-
pholipids in sponge cellular membranes. A high abundance of these
may reflect physiological differences that could facilitate the resis-
tance of these species to environmental stress (Koopmans et al.,
2015; Lawson et al., 1988; Mueller-Navarra, 1995; Tocher, 2003).
The presence of n-3 and n-6 LC PUFA is consistently associated
with increased stress resistance across a range of taxa, with deficien-
cies correlated with reduced growth, as well as increased mortality
and susceptibility to stressors and disease (Bachok, Mfilinge, & Tsu-
chiya, 2006; Immanuel, Palavesam, & Petermarian, 2001; Koven
et al., 2001; M€
uller-Navarra, Brett, Liston, & Goldman, 2000; Parrish,
2013; Pernet & Tremblay, 2004). An abundance of bioactive PUFA
would provide buffering capacity for sponges under stressful condi-
tions, and thus contribute to the ability of these sponges to maintain
membrane function and subsequent cell homeostasis in the face of
environmental change. S. flabelliformis had a particularly high content
of ARA, a FA involved in eicosanoid synthesis. Eicosanoids are
metabolites that play a role in cellular regulation of processes such
as the fluid and electrolyte fluxes that are important in the regula-
tion of membranes, e.g. during a thermal stress response (Koven
et al., 2001).
The low overall PUFA content of C. coralliophila may also explain
why this species is better able to tolerate increased temperature,
FIGURE 5 Exploration of potential HVA methods in sponge holobionts. The effects of OW and OA on: (a) the ratio of sterol to
phospholipid; (b) the ratio of structural to storage lipid; (c) Mean chain length (MCL); (d) the ratio of SFA to PUFA; and (e) the degree of FA
unsaturation (DoU) for each species exposed to OW and OA (n=3 to 6 individuals per species per treatment, except where mortalities
occurred for Rhopaloeides odorabile where n=2 at 31.5°C/pH 7.6). P =phototrophic species and H =heterotrophic species
10
|
BENNETT ET AL.
compared to the sensitive phototrophic C. foliascens. While n-3 and
n-6 LC PUFA are important for stress resistance, PUFA of thylakoid
membranes in photosynthetic organisms are particularly sensitive to
temperature and are the primary targets of lipid oxidation (Boti
c
et al., 2015; Wada & Murata, 1998; Wada et al., 1994). Therefore
high PUFA concentrations may put C. foliascens at an elevated risk
of lipid oxidation, which may, at least partially, explain its sensitivity
to OW. Further work exploring lipid oxidation in these species upon
exposure to OW would help elucidate how a high PUFA content
impacts the ability of sponges to tolerate thermal stress.
4.2
|
Sponge lipid and FA profiles in a high CO
2
world
While OW and OA did not significantly impact sponge lipid profiles,
the FA profile of all species, except S. flabelliformis, shifted signifi-
cantly with increasing temperature. Bleached phototrophic sponges
(characterized by reduced chlorophyll acontent) experienced signifi-
cant declines in their dominant SFA (16:0), whereas the concentra-
tion of the second most abundant SFA in these two species (22:0
for C. foliascens and 14:0 for C. coralliophila) increased with tempera-
ture. The decline in 16:0 in thermally stressed phototrophic sponges
is either due to loss of symbionts or reflects a breakdown in sym-
biont FA biosynthesis, and the subsequent reduced translocation of
this FA to the sponge host (Figueiredo et al., 2012; Hillyer, Tuma-
nov, Villas-B^
oas, & Davy, 2016; Imbs & Yakovleva, 2012). Mean-
while, the increase in other abundant SFA either reflects a switch in
diet, e.g. to acquiring carbon heterotrophically from the water col-
umn to compensate for reduced photosynthate translocation during
bleaching (Grottoli, Rodrigues, & Juarez, 2004; Grottoli, Rodrigues, &
Palardy, 2006; Hoadley et al., 2015), or a different metabolic
response by the host to replace lost 16:0. The concentration of the
short chain SFA 6:0 also increased with temperature for both spe-
cies. Short chain SFA tend to be of bacterial origin (Berg
e & Bar-
nathan, 2005), suggesting an increase in microbial abundance, and
associated short chain SFA neogenesis, in bleached sponges. The
MUFA characteristic of healthy sponges declined in bleached pho-
totrophic sponges. MUFA are a readily catabolized energy source
(Tocher, 2003) and it is possible that bleached sponges break down
these MUFA to produce energy as compensation for the energy def-
icit that occurs with photosynthetic dysfunction.
Likewise, MUFA characteristic of T0 sponges declined with tem-
perature in the sensitive heterotrophic R. odorabile, again suggesting
catabolism of these MUFA by the sponge host. R. odorabile has an
abundance of nonphotosynthetic microbes (Webster & Hill, 2001;
Webster, Wilson, Blackall, & Hill, 2001) and temperature-related
mortality has previously been correlated with a breakdown in the
relationship between the host and its associated symbionts (Fan, Liu,
Simister, Webster, & Thomas, 2013; Webste, Cobb, & Negri, 2008)
which, like phototrophic symbionts, play important metabolic roles in
the symbiosis. R. odorabile feeding efficiency is also reduced under
thermal stress (Massaro, Weisz, Hill, & Webster, 2012), and this,
combined with symbiosis breakdown, suggests that this species also
catabolizes MUFA to generate energy due to its typical energy
source being compromised. Interestingly, however, the overall
R. odorabile MUFA concentration increased due to a doubling of 17-
Me 26:1 n-9 (a “demospongic acid”). The relative proportion of such
FA in sponges has been shown to vary with season, and is suggested
to play a role in the maintenance of sponge membrane fluidity with
changes in temperature (Hahn et al., 1988). An increase in this FA
may act to reduce the fluidity of thermally-perturbed cell mem-
branes, possibly reflecting a thermal stress response by these
sponges (Guerzoni et al., 2001; Hochachka & Somero, 1984).
Sponge PUFA content increased in both phototrophic species
with increasing temperature. For the sensitive C. foliascens, this
increase occurred alongside a significant shift in LC PUFA concentra-
tion. While the concentration of DPA (22:5 n-3) increased, that of
EPA (20:5 n-3) declined by almost half compared to sponges
exposed to 28.5°C, indicating that EPA is elongated to DPA, which
is particularly important for the structural integrity of cell membranes
(Anholt, 2004). The concentration of another important FA, ARA
(20:4n-6), declined by almost half when both thermally sensitive spe-
cies were exposed to 31.5°C, compared to sponges exposed to
28.5°C. It is possible that this decline reflects ARA entering the eico-
sanoid pathway, a stress-signalling cascade activated by exposure of
cells to oxidative stress (Hillyer et al., 2016; L~
ohelaid, Teder, &
Samel, 2015). Meanwhile, the more thermally tolerant species expe-
rienced no change in LC PUFA concentration. The stable PUFA con-
tent of these sponges under elevated temperature reflects the
tolerance of these sponges to thermal stress, and indicates either
selective retention of LC PUFA under stress or simply an absence of
stress associated with such conditions for these species. Regardless,
depletion of these important PUFA, as observed in the sensitive
sponge species, is commonly associated with a deterioration in
organism health, as evidenced in the bleached coral Pavona frondifera
for which a reduction in n-3 and n-6 PUFA increased susceptibility
to disease and mortality (Bachok et al., 2006).
4.3
|
Homeoviscous adaptation
We explored the ability of sponges to compositionally alter their
membrane lipids to prevent membrane destabilization in response to
environmental stress using a number of different HVA indicators.
The ratio of sterols to phospholipids increased significantly at
reduced pH for the thermally sensitive C. foliascens, with the great-
est increase occurring in the high OW/OA treatment. Sterols and
phospholipids are the primary constituents of the lipid bilayer, and
sterols are particularly important for maintaining membrane rigidity
with changing environmental conditions (Los & Murata, 2004; Par-
ent, Pernet, Tremblay, Sevigny, & Ouellette, 2008; Presti, 1985); for
instance bivalve species living in variable environments, such as sur-
face waters, alter the sterol concentration of their cell membranes in
response to seasonally varying temperatures (Copeman & Parrish,
2004; Parrish, 2013). Additional inorganic carbon available under OA
may stimulate symbiont photosynthetic rates, resulting in an
increased translocation of photosynthetically derived carbon to
BENNETT ET AL.
|
11
C. foliascens (Fu, Warner, Zhang, Feng, & Hutchins, 2007; Morrow
et al., 2015). It appears that C. foliascens is able to utilize this addi-
tional carbon to increase sterol biosynthesis, likely reflecting a HVA
mechanism and providing a putative pathway via which elevated
CO
2
facilitates resistance to thermal stress.
The average FA chain length increased significantly with temper-
ature for R. odorabile (primarily through an increase in the concentra-
tion of the demospongic acid 17-Me 26:1 n-9), likely reflecting an
attempt at membrane stabilization by increasing the proportion of
FA with higher melting points (Suutari & Laakso, 1994).
The degree of FA unsaturation (DoU) increased significantly with
temperature for both phototrophic species, yet decreased signifi-
cantly for the heterotrophic R. odorabile with OW, and although
nonsignificant, the DoU in S. flabelliformis was lowest in the high
OW/OA treatment. A decrease in DoU suggests either an attempt
by the heterotrophic sponge to mitigate peroxidation, or more likely,
increased host metabolism in response to stress (Hillyer et al., 2016).
An increase in the proportion of unsaturated FA, on the other hand,
has been observed for a number of photosynthetic microorganisms
in response to super-optimal temperatures (Guerzoni, Ferruzzi, Sini-
gaglia, & Criscuoli, 1997; Guerzoni et al., 2001; Guillot et al., 2000;
Wada et al., 1994). This is due to the activation of the oxygen-
dependent desaturase system, which not only introduces double
bonds into SFA to increase DoU, but in turn protects cells from
oxidative and thermal stress by consuming additional oxygen and
reactive oxygen species accumulated at high temperatures (Guerzoni
et al., 1997, 2001). This is supported by observed declines in the
ratio of SFA to PUFA for both phototrophic sponges with increased
temperature (although nonsignificant for C. foliascens) and an under-
standing of the cellular oxidation levels experienced by these species
following exposure to OW would help confirm this stress response
pathway. It is important to note that the lipid and FA profiles pre-
sented in this study are for the sponge holobiont; i.e. the sponge
animal and its associated symbionts in their entirety. It is probable
that the difference in response between these two nutritional types
is the culmination of both the animal and microbial components of
the sponge holobiont responding differently to thermal stress. Future
work exploring the sponge host and associated symbiont responses
in isolation is therefore required to better understand the mecha-
nisms underlying these observed responses. Nevertheless, activating
an oxygen-consuming desaturase system would be particularly
important for phototrophic organisms, due to the excessive produc-
tion of reactive oxygen species as a result of the inactivation of the
oxygen-evolving capability of PSII at stressful/photoinhibiting tem-
peratures (Wada & Murata, 1998).
5
|
CONCLUSION
Through the exploration of sponge lipid and FA composition we
reveal previously uncharacterized components in the sponge stress
response, providing insight into potential mechanisms contributing to
the resilience of this ecologically important phylum during
environmental change. Sponges with a greater content of storage
lipids, as well as a higher proportion of phospholipids and sterols,
and higher concentrations of n-3 and n-6 PUFA exhibited the great-
est resistance to OW and OA. These lipids are the primary con-
stituents of the lipid bilayer of cell membranes and likely enable
sponges to maintain membrane function and cell homeostasis in the
face of environmental change (Geider, 1987; Guillot et al., 2000;
Hazel, 1995; Singh et al., 2002), including OW and OA as demon-
strated here. We also reveal that sponges can respond to thermal
perturbations with a diversity of lipid and FA alterations, including
shifting the proportion of membrane lipids, and changing the degree
of FA unsaturation and FA elongation. Such mechanisms likely con-
tribute to the acclimatization potential of these species under cli-
mate change, although direct measurements of membrane fluidity
would further substantiate this stress response pathway. Finally, we
discovered distinct differences in the responses of phototrophic and
heterotrophic sponges to thermal stress, suggesting that associated
photosymbionts and the sponge host respond differently.
ACKNOWLEDGEMENTS
This project was funded by the Australian Institute of Marine
Science, Victoria University of Wellington, the PADI foundation, the
Royal Society of New Zealand Marsden Fund VUW1505 and Deakin
University. NSW was funded through an Australian Research Council
Future Fellowship FT120100480, HMB was funded by a VUW Doc-
toral Scholarship. Staff of the Australian Institute of Marine Science,
particularly SeaSim staff, Christine Altenrath, Florita Flores, Andrew
Negri, Stephen Boyle and Sarah Sutcliffe (JCU student volunteer) are
acknowledged for their invaluable assistance in running the experi-
ment in the Sea Simulator. Jessica Conlan and Julia Strahl are
acknowledged for sharing their expertise.
ORCID
Holly Bennett http://orcid.org/0000-0002-4085-8023
James J. Bell http://orcid.org/0000-0001-9996-945X
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How to cite this article: Bennett H, Bell JJ, Davy SK,
Webster NS, Francis DS. Elucidating the sponge stress
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