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Trophic Ecology of the Tropical Pacific Sponge Mycale grandis Inferred from Amino Acid Compound-Specific Isotopic Analyses

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Many sponges host abundant and active microbial communities that may play a role in the uptake of dissolved organic matter (DOM) by the sponge holobiont, although the mechanism of DOM uptake and metabolism is uncertain. Bulk and compound-specific isotopic analysis of whole sponge, isolated sponge cells, and isolated symbiotic microbial cells of the shallow water tropical Pacific sponge Mycale grandis were used to elucidate the trophic relationships between the host sponge and its associated microbial community. δ¹⁵N and δ¹³C values of amino acids in M. grandis isolated sponge cells are not different from those of its bacterial symbionts. Consequently, there is no difference in trophic position of the sponge and its symbiotic microbes indicating that M. grandis sponge cell isolates do not display amino acid isotopic characteristics typical of metazoan feeding. Furthermore, both the isolated microbial and sponge cell fractions were characterized by a similarly high ΣV value—a measure of bacterial-re-synthesis of organic matter calculated from the sum of variance among individual δ¹⁵N values of trophic amino acids. These high ΣV values observed in the sponge suggest that M. grandis is not reliant on translocated photosynthate from photosymbionts or feeding on water column picoplankton, but obtains nutrition through the uptake of amino acids of bacterial origin. Our results suggest that direct assimilation of bacterially synthesized amino acids from its symbionts, either in a manner similar to translocation observed in the coral holobiont or through phagotrophic feeding, is an important if not primary pathway of amino acid acquisition for M. grandis.
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HOST MICROBE INTERACTIONS
Trophic Ecology of the Tropical Pacific Sponge Mycale grandis
Inferred from Amino Acid Compound-Specific Isotopic Analyses
Joy L. Shih
1
&Karen E. Selph
1
&Christopher B. Wall
2
&Natalie J. Wallsgrove
3
&Michael P. Lesser
4
&Brian N. Popp
3
Received: 19 March 2019 /Accepted: 2 July 2019
#Springer Science+Business Media, LLC, part of Springer Nature 2019
Abstract
Many sponges host abundant and active microbial communities that may play a role in the uptake of dissolved organic matter
(DOM) by the sponge holobiont, although the mechanism of DOM uptake and metabolism is uncertain. Bulk and compound-
specific isotopic analysis of whole sponge, isolated sponge cells, and isolated symbiotic microbial cells of the shallow water tropical
Pacific sponge Mycale grandis were used to elucidate the trophic relationships between the host sponge and its associated microbial
community. δ
15
Nandδ
13
CvaluesofaminoacidsinM.grandis isolated sponge cells are not different from those of its bacterial
symbionts. Consequently, there is no difference in trophic position of the sponge and its symbiotic microbes indicating that M.
grandis sponge cell isolates do not display amino acid isotopic characteristics typical of metazoan feeding. Furthermore, both the
isolated microbial and sponge cell fractions were characterized by a similarly high ΣVvaluea measure of bacterial-re-synthesis of
organic matter calculated from the sum of variance among individual δ
15
N values of trophic amino acids. These high ΣVvalues
observed in the sponge suggest that M.grandis is not reliant on translocated photosynthate from photosymbionts or feeding on water
column picoplankton, but obtains nutrition through the uptake of amino acids of bacterial origin. Our results suggest that direct
assimilation of bacterially synthesized amino acids from its symbionts, either in a manner similar to translocation observed in the
coral holobiont or through phagotrophic feeding, is an important if not primary pathway of amino acid acquisition for M.grandis.
Keywords Dissolved organic matter .Symbioses .Amino acid translocation .δ
13
C.δ
15
N
Introduction
Sponges are ubiquitous members of benthic coral reef com-
munities and many species are known to host diverse and
metabolically active symbiotic microbial communities [1].
Sponges are essential to sustaining high secondary
productivity on oligotrophic coral reefs through their rapid
uptake of dissolved organic matter (DOM) and subsequent
shedding of particulate organic matter in the form of their
choanocyte cells. This sponge detritus is then available to a
variety of benthic detritivores [2], driving an efficient trans-
fer of DOM to higher trophic levels via the sponge-loop
pathway [3,4].
The mechanisms by which DOM is assimilated and trans-
ferred by the sponge is not well known but may be facilitated
by a close symbiosis between sponges and their microbial
communities [2,512]. This symbiosis is often functionally
mutualistic and is among the most ancient of metazoan sym-
bioses [13]. The sponge-microbe symbiosis is fundamental to
sponge ecology, as even distantly related sponges from geo-
graphically disparate regions share a common set of associat-
ed microbes, some of which are unique to sponges and cannot
be acquired from the surrounding environment [1419].
Sponge-associated symbionts also contribute to the carbon
(C) and nitrogen (N) requirements of sponges [10,20], and
some sponge symbionts produce secondary metabolites that
provide for their hosts chemical defenses [2124].
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s00248-019-01410-x) contains supplementary
material, which is available to authorized users.
*Brian N. Popp
popp@hawaii.edu
1
Department of Oceanography, University of HawaiiatMānoa,
Honolulu, HI 96822, USA
2
Hawaii Institute of Marine Biology, University of HawaiiatMānoa,
PO Box 1346, Kāneohe, HI 96744, USA
3
Department of Earth Sciences, University of HawaiiatMānoa,
Honolulu, HI 96822, USA
4
Department of Molecular, Cellular and Biomedical Sciences,
University of New Hampshire, Durham, NH 03824, USA
Microbial Ecology
https://doi.org/10.1007/s00248-019-01410-x
The invasive sponge Mycale grandis is found in Kāne ohe
Bay and other partially degraded coral reef habitats around the
main Hawaiian Islands. It is unknown whether M.grandis
obtains C and N from its microbial symbionts in addition to,
or instead of, heterotrophic filter feeding. Some sponges are
able to obtain half of their energy budget and more than half of
their C budget from their photosymbionts [25], whereas DOM
can comprise 90% of the diet in other species [7,9,10,26].
For instance, shallow water Caribbean sponge species derive
much of their required metabolic C from dissolved organic
carbon (DOC), while Indo-Pacific species rely more heavily
on photoautotrophy [25,27]. Since many sponges harbor over
10
9
bacterial cells g
1
of sponge wet weight [16], we investi-
gated whether bacterially derived DOM may be a significant
source of DOM to sponges.
The main producers of DOM on coral reefs are primary
producers, particularly macroalgae and the endosymbiotic di-
noflagellates (Family Symbiodiniaceae) [28] of scleractinian
corals, which directly or indirectly release a substantial frac-
tion of their excess photosynthate into the water column [29].
As a result, early studies sought to investigate specific sources
of DOM available to sponges. de Goeij et al. [26]used
13
C-
enriched diatom DOM and particulate organic matter (POM)
to positively demonstrate that algal-derived DOC was assim-
ilated by the sponge and furthermore shed as sponge cells
(e.g., choanocytes), and this detritus was consumed by other
reef fauna at higher trophic levels. Rix et al. [12]used
13
C- and
15
N-labeled warm and cold-water coral mucus DOM as food
sources for a warm-water Red Sea sponge and a North
Atlantic cold-water sponge, respectively, and demonstrated
assimilation of coral mucus into sponge tissue and subsequent
sponge-derived detritus production. Subsequently, studies
using
13
C-labeled DOM have shown
13
C uptake of coral-
derived DOM by sponge cells and algal-derived DOM by
the symbiotic microbes [8]. Although the pathways of acqui-
sition of DOM may vary across species, DOM from the two
sources were shown to be assimilated by the sponge and sub-
sequently released as sponge-derived detritus; however, algal-
derived DOM was released as sponge cell detritus at a higher
rate. Sponge cells, however, are probably capable of taking up
the larger colloidal fraction in DOM such as viruses and free
amino acids, while bacteria can assimilate the smaller, truly
dissolved fraction of DOM [26,30,31].
The study of diverse and complex consortia of symbionts
within sponges is challenging because multiple functions and
interactions can take place simultaneously and it is difficult to
resolve the specific roles and contribution of the symbionts
and host. We use stable C and N isotope analyses of whole
sponge, sponge cell, and microbial cell fractions, as well as
isotopic analyses of individual amino acids (AAs) in each cell
fraction to elucidate the trophic pathways utilized by M.
grandis. Carbon isotopic composition (δ
13
C values) is used
to determine the sources of an organisms dietary C. Nitrogen
isotopic composition (δ
15
Nvalues)hasbeenusedinearlier
studies of marine food webs as an indicator of relative trophic
position of an organism based on the consistent increase in
15
N/
14
N ratios from food source to consumer [32,33].
However, understanding key dietary details can be limited
due to multiple factors influencing bulk tissue δ
13
Cand
δ
15
N values, including differences in baseline values and un-
certainty regarding the magnitude of trophic discrimination
factors of organisms.
Compound-specific isotopic measurements of AAs can be
used to address some of these challenges. δ
15
Nvaluesofin-
dividual AAs allows for determination of trophic positions
(TP) based on the differential fractionation of AAs from food
source to consumer. In samples of consumer tissues, source
AAs retain the isotopic composition of N sources at the base
of the food web, whereas trophicAAs are greatly enriched
in
15
N relative to source AAs with each trophic transfer
[3437]. The utility of the amino acid compound-specific iso-
topic analysis (AA-CSIA) approach is that the N isotopic
composition of an individual AA reflects the degree of isoto-
pic fractionation associated with various biochemical reac-
tions that involve C-N bond breakage (deamination and trans-
amination) in individual AAs involved in N metabolism.
However, the exact biochemical mechanisms are complex
[38,39]. The δ
15
N value of an organism also inherently re-
flects the isotopic composition of inorganic N sources (nitrate,
nitrite, ammonia, and urea) assimilated by primary producers
at the base of the food web. Since source AAs retain δ
15
N
values from the base of the food web, AA-CSIA provides
information about N metabolism, constrain TP, and identify
the source and transformation of dissolved and detrital organic
matter in marine waters and sediments [4042]. Similar to
δ
15
NvaluesofsourceAAs,theδ
13
C values of essential amino
acids (EAA) in consumers retain the carbon isotopic compo-
sition of the plants and bacteria that produced those AAs [43].
Here, we provide experimental evidence that the means of
transferring nutrition from microbial symbiont to the sponge
M.grandis is through the uptake of AAs synthesized by its
symbiotic bacteria, and bacterially produced AAs are a major
substrate by which C and N are transferred from microbial
symbiont to the sponge. The efficiency of this transfer mech-
anism is facilitated by the high concentrations of microbial
communities hosted within the sponge mesohyl.
Methods
Sampling Locations
Kāne ohe Bay on the northeast coast of O ahu, Hawaii, is
characterized by an extensive system of scleractinian coral
dominated fringing reefs, patch reefs, and a large barrier reef.
Sponge samples for bulk tissue isotopic analysis were
Shih J. L. et al.
collected on May 6, 2015 from two patch reefs just northwest
of Coconut Island (21° 2607.5′′ N157°4743.9′′ W) in the
southern portion of Kāne ohe Bay (n= 34), from a patch reef
in mid Kāne ohe Bay (21° 2737.5′′ N157°4919.7′′ W) (n=
10), and from a patch reef in the northern portion of Kāne ohe
Bay (21° 2837.6′′ N 157° 4954.7′′ W) (n= 10). Sponge
samples for bulk and compound-specific isotopic analysis of
isolated cell fractions (n= 8) were collected from ~ 1 m depth
on May 3, 2017 from the fringing reef located beneath the
Lilipuna Pier in south Kāne ohe Bay (21° 2546.0′′ N157°
4731.2′′ W). Sponge specimens were inclusive of surface
pinacoderm, inner mesohyl, and choanocyte chambers.
Visible debris and epiphytes were removed from the surface
of the samples. Specimens from north, mid, and south
Kāne ohe Bay collected for bulk isotopic analysis were imme-
diately frozen at 80 °C and stored until analysis. Whole
sponge samples for epi-fluorescence microscopy were collect-
ed from under the Lilipuna Pier, rinsed with filtered seawater,
and preserved in 1% paraformaldehyde. Isotope values for
planktonic end members were determined using oblique net
tows (63 μm mesh) on January 19, 2018, west of Coconut
Island and east of Lilipuna Pier (21° 2548.5′′ N157°4727.0
v W). Plankton were size-fractioned using nylon mesh be-
tween 63 and 250 μm and consisted predominantly of cope-
pods and crab zoea. Plankton were concentrated on a GF/F
filter (0.7 μm nominal poe size), rinsed with distilled H
2
O,
dried (60 °C) and ground to a powder, and stored until AA-
CSIA. While these samples only contained zooplankton, δ
15
N
values of source AAs and δ
13
C values of EAA will reflect that
of the phytoplanktonic end member.
Separation of Microbial Cells and Sponge Cells
from Sponge Tissue
Sponge and microbial cells of M.grandis were separated
through size fractionation using modification of the methods
described in Freeman and Thacker [44,45]. Samples were
homogenized with mortar and pestle in filtered seawater to
dissociate the sponge cells and vacuum filtered (Whatman
No. 4 filter, 2025 μm nominal pore size). The resulting fil-
trate was centrifuged (430×g) for 6 min to form a sponge cell
pellet. The supernatant containing microbial cells was
decanted and stored at 20 °C. To rinse the sponge cell pellet,
it was suspended in filtered seawater and centrifuged (twice,
5 min, 4 °C, 770×g). Further rinsing was accomplished by
suspension and centrifugation (twice, 2 min, 3900×g). The
isolated sponge cell pellet was stored frozen (20 °C) until
analysis.
To isolate microbial cells, the previously frozen superna-
tant containing the microbial fraction was thawed and centri-
fuged (17,00g) for 17 min at 4 °C. The supernatant was
decanted, the pellet resuspended, and the centrifuge step re-
peated. The pellet was resuspended and transferred to a
1.5 mL centrifuge tube and the microbial cell fraction centri-
fuged (12,800×g) for 2 min, and the supernatant was carefully
removed and discarded. The resulting microbial pellet was
rinsed twice using the same procedure and the remaining pel-
let was frozen at 20 °C until analysis.
Cell Abundance and Identification (Flow Cytometry,
Microscopy)
A small volume (40 μL) of the separated sponge cell and
microbial cell fractions were fixed in paraformaldehyde (1%
final concentration), and frozen (80 °C) until batch analyses
for cell abundances using flow cytometry. The flow cytometer
was a Beckman Coulter EPICS Altra flow cytometer with a
Harvard Apparatus syringe pump for volumetric sample de-
livery. Simultaneous (co-linear) excitation of the cells was
provided by two water-cooled 5-W argon ion lasers tuned to
488 nm (1 W) and the UV range (200 mW). Samples were
thawed and diluted with 800 μL of filtered seawater and
stained with the DNA-specific stain Hoechst 33342
(1 μgmL
1
final concentration) for 1 h in the dark. Aliquots
of 100 μL were analyzed for Synechococcus (SYN), photo-
synthetic eukaryotes, and non-pigmented bacterial abun-
dances. Discrete populations were enumerated on the basis
of chlorophyll a(red fluorescence, 680 nm), phycoerythrin
(orange fluorescence, 575 nm), DNA (blue fluorescence,
450 nm), forward scatter, and 90° scatter signatures.
Listmode files were processed using FlowJo software
(Treestar Inc., www.flowjo.com). The DNA fluorescence
detector was set at 700 V for the microbial samples, whereas
the sponge cell samples were analyzed at a much lower
voltage (400 V) to eliminate microbial cells from the gathered
data files.
Cell abundances were converted to carbon for each popu-
lation, assuming 30 fg cell
1
for bacteria [46], 200 fg cell
1
for
Synechococcus and other picophytoplankton, 800 fg cell
1
for
nanophytoplankton [47], and 3 pg cell
1
for sponge cells [48].
Epi-fluorescence microscopy (Olympus BX-41) was used
to examine sponge cell and microbial isolates. Preserved sam-
ples were thawed, stained with 4,6-diamidino-2-phenylindole
(1 μgmL
1
final concentration) and proflavin 3,6-
diaminoacridine, hemisulfate salt for protein-staining (0.33%
w/v, final concentration), then filtered onto black 0.8 μm
25 mm PCTE filters and mounted on glass slides. Samples
were observed (×200 and × 400) under blue and UV excita-
tion wavelengths, to determine pigment (chlorophyll, phyco-
erythrin) presence and observe DNA contents.
Bulk Sponge and Cell Isotope Analysis
The δ
13
Candδ
15
N values of lyophilized bulk sponge tissue
(1.72.1 mg), isolated sponge (0.20.3 mg), and microbial
cells (0.30.4 mg) were determined using a Costech elemental
Trophic Ecology of the Tropical Pacific Sponge Mycale grandis Inferred from Amino Acid Compound-Specific...
combustion system (Model 4010) coupled to a
ThermoFinnigan Delta Plus XP isotope ratio mass spectrom-
eter (IRMS) through a Conflo IV interface. Isotopic composi-
tions are reported in typical δ-notation relative to the interna-
tionally recognized standards V-PDB and atmospheric AIR
,
respectively. Accuracy and precision were < 0.2,asdeter-
mined from multiple laboratory reference materials extensive-
ly calibrated using National Institute of Science and
Technology reference materials and analyzed every 10
samples.
Preparation of Samples for Amino Acid Isotope
Analysis
Isolated sponge, microbial cells, and plankton were prepared
for compound-specific amino acid δ
13
Candδ
15
N analysis.
Due to insufficient materials, cell fractions isolated from sep-
arate sponges were combined prior to analysis. Individual
sponge samples are designated sponge n, whereas sponge
and microbial cell isolates are Snand Mnrespectively, where
nrepresents the sponge genotype. Microbial fractions for
sponges 1 and 3 were combined, as were the sponge cell
fractions. For sponges 4, 6, and 7, sufficient materials were
available for AA-CSIA of microbial cell fractions for each
sponge; however, these three sponge cell isolates were com-
bined for δ
13
Candδ
15
Nanalysis.
Samples for AA-CSIA were hydrolyzed and
trifluoroacetyl/isopropyl ester derivatives created according
to the methods of Popp et al. [36] and Hannides et al. [49].
Samples (411 mg) were hydrolyzed (trace-metal grade 6 M
HCl, 150 °C, 70 min) and the hydrolysate purified using low
protein-binding filters and cation exchange chromatography.
Purified samples were esterified using 4:1 isopropanol:acetyl
chloride and derivatized using 3:1 methylene
chloride:trifluoroacetyl anhydride. Trifluoroacetyl/isopropyl
ester derivatives were additionally purified using solvent ex-
traction [50] and stored at 20 °C for up to 2 weeks before
analysis. Samples were prepared with an additional vial con-
taining a mixture of 15 pure AAs purchased commercially
(Sigma Scientific).
Nitrogen Isotope Analysis of Amino Acids
The δ
15
N values of AA trifluoroacetyl/isopropyl ester deriva-
tives were determined using gas chromatography combustion
isotope ratio mass spectrometry (GC-C-IRMS, [51]). The iso-
tope ratio mass spectrometer (IRMS; Thermo Scientific Delta
V) was interfaced to a gas chromatograph (GC; Thermo
Scientific Trace) fitted with a 60 m BPX5 forte column
(0.32 mm internal diameter with 1.0 μm film thickness;
SGE, Inc.) through a GC-C III combustion furnace (980 °C),
reduction furnace (650 °C), and liquid nitrogen cold trap.
Helium (1.2 mL min
1
) was used as the carrier gas. Prior to
analysis, samples were dried and redissolved in an appropriate
volume of ethyl acetate. Each sample was analyzed in at least
triplicate, with norleucine and aminoadipic acid internal refer-
ence compounds co-injected in each run. The suite of 15 pure
amino acids was also analyzed every 3 injections to provide an
additional measure of instrument accuracy. The δ
15
Nvalues
of all pure amino acid reference compounds were previously
determined using the bulk isotope technique described above.
Nitrogen isotope values are reported in standard δ-notation
relative to atmospheric AIR. For replicate injections of sam-
ples, amino acid δ
15
N standard deviations averaged 0.5and
ranged from 0.1 to 1.0.
Carbon Isotope Analysis of Amino Acids
δ
13
C values of individual amino acid trifluoroacetyl/isopropyl
ester derivatives were determined using an IRMS (MAT 253)
interfaced with a Trace GC Ultra via a combustion furnace
(1000 °C) and ConFlo IV interface (Thermo Scientific).
Samples were injected using a PTV (pressure/temperature/
volume) injector, held at 40 °C for 3 s, heated to 87 °C
(400 °C min
1
), heated again to 200 °C and transferred at
20Cusinga1:10split.Helium(1mLmin
1
) was used as
the carrier gas. The gas chromatograph was fitted with a BPX5
forte capillary column (30 m × 0.32 mm internal diameter
with 1.0 μm film thickness; SGE, Inc.). The oven temperature
for the GC started at 40 °C and was held for 1 min before
heating at 15 °C min
1
to 120 °C, then 3 °C min
1
to 190 °C,
and finally 5 °C min
1
to 300 °C where it was held for an
additional 10 min. Isotope values are reported in standard δ-
notation relative to V-PDB. Each sample was analyzed in at
least triplicate with a perdeuterated n-C
20
alkane with a well-
characterized δ
13
C value co-injected as an internal reference.
The 15 AA reference suite was analyzed every three injec-
tions, and sample δ
13
C
AA
values corrected relative to this
AA suite following Silfer et al. [52]. For statistical analysis,
δ
13
C
AA
values were compared to those previously published
by Larsen et al. [43,53,54]. To account for inter-laboratory
differences, corrections based on results of previous extensive
calibrations were used [55,56].
Trophic Proxy and Trophic Position
Trophic position was estimated using the difference in δ
15
N
values between the trophic amino acid glutamic acid (Glx) and
the source amino acid phenylalanine (Phe). This calculation
assumed a βvalue of 3.4for the difference in δ
15
Nvalues
between Glx and Phe in primary producers and assumed that
Glx was enriched in
15
N relative to Phe (Δvalue) by 7.6
with each trophic transfer, e.g.: TP = ((δ
15
N
Glx
δ
15
N
Phe
3.4) / 7.6) + 1 [34]. Uncertainty in calculations of trophic po-
sition was determined using propagation of errors [57,58].
The propagated error in this TP calculation assumed the
Shih J. L. et al.
uncertainty in βvalue is ± 0.9and the error in Δis ± 1.1
and used the measured analytical uncertainty in δ
15
N
Glx
and
δ
15
N
Phe
based on at least triplicate analyses.
A proxy for trophic position was also determined using the
difference in averaged δ
15
N values between trophic (Alanine
[Ala], Leucine [Leu], Glx) and source amino acids (Lysine
[Lys], Phe). A second trophic position proxy was also calcu-
lated using weighted mean δ
15
N values of trophic (Ala, Leu,
Glx) and source amino acids (Lys, Phe) using:
δ15N
xw
¼
δ15Nx
σ2
x
1
σ2
x
;ð1Þ
where δ
15
N
x
is the nitrogen isotopic composition of a speci-
fied AA and σ
x
is the standard deviation of the δ
15
Nvalueof
that AA determined by triplicate isotopic measurements [51,
57]. Errors for proxy trophic position calculations were prop-
agated using the measured reproducibility of individual AA in
at least triplicate.
Summed Variance in δ
15
N Values of Trophic Amino
Acids (ΣV)
McCarthy et al. [40] introduced an index for microbial re-
synthesis of amino acids: ΣV, which is the sum of variance
among individual δ
15
N values of trophic amino acids. ΣV
values for isolated sponge cell and microbial cell fractions
were calculated using δ
15
NvaluesofAla,Leu,Proline
(Pro), Aspartic acid (Asx), and Glx.
Data Analysis
Statistical tests were conducted using SigmaPlot (v.13,
Systat Software) and R [59]. For all statistical analyses,
we considered pvalues < 0.05 statistically significant.
Statistical differences between geographic regions in
Kāne ohe Bay were tested using one-way analysis of var-
iance tests (ANOVAs). Data distributions were assessed
using histograms, with log-transformations used to im-
prove normality when necessary. When ANOVA results
were significant, a post-hoc Tukeys honest significant dif-
ference (HSD) test was conducted to determine which
groups differed from others. Hierarchical cluster analysis
of the δ
13
C values of essential amino acids and δ
15
Nvalues
of source amino acids of microbial cells, sponge cells, and
plankton were performed in R using scaled data in a
Euclidian dissimilarity matrix with the Ward clustering cri-
terion. Clusters were validated using silhouette plots in the
package factoextra [60].
Results
Flow Cytometry
In the sponge cell samples, two sponge cell populations were
observed with flow cytometry: cells with very high DNA and
chlorophyll fluorescence (Sponge-1), which were most likely
sponge cells containing phytoplankton prey, and sponge cells
without prey or with non-photosynthetic bacterial prey in
them (Sponge-2, low DNA, and chlorophyll fluorescence
per sponge cell). Both populations had high light scatter in-
dicative of larger cells (Online Resource Figure 1). There were
ca. six times more sponge cells present than residual phyto-
plankton or bacterial cells in these samples (Online Resource
Tab le 1). The carbon contribution of sponge cells averaged 94
± 4% (range 85% to 98%), while the residual microbial cell C
ranged from 1.8 to 15% and averaged 6.3 ± 4% (Table 1).
Sponge cell fractions contained a mean of 6 × 10
3
cells g
1
wet weight (range 1.4 to 12.1 × 10
3
cells g
1
,n=8, Online
Resource Table 1). Sponge-1 cell counts averaged 2 × 10
3
cells g
1
, while Sponge-2 cell counts averaged 4 × 10
3
cells
g
1
. Residual microbial cells in these samples had a mean of
1×10
3
cells g
1
wet weight, with approximately equal cell
contributions (both ~ 0.5 × 10
3
cells g
1
) of Microbe-1 (small-
er phytoplankton and bacterial cells) and Microbe-2 (larger
phytoplankton).
Table 1 Composition (percent of total) of microbial (M) and sponge (S)
cell fractions by carbon contribution, after determination of cell abun-
dances by flow cytometry. Populations shown are non-pigmented, het-
erotrophic bacteria (HBACT), phytoplankton without Synechococcus
(PHYTO), Synechococcus (SYN), and sponge cells (SPONGE).
Populations determined as described in the text
Sample HBACT PHYTO SYN Sponge
M1 79 21 0 0
M2 77 23 0 0
M3 33 66 1 0
M4 84 16 0 0
M5 41 58 1 0
M6 82 18 0 0
M7 83 17 0 0
M8 73 26 1 0
S1 8 0 0 92
S2 7 0 0 93
S3 16 0 0 84
S4 2 0 0 98
S5 7 0 0 93
S6 4 0 0 96
S7 3 0 0 97
S8 4 0 0 96
Trophic Ecology of the Tropical Pacific Sponge Mycale grandis Inferred from Amino Acid Compound-Specific...
The sponge cell fraction samples S6 through S8 contained
significantly more Sponge-2 cells (p= 0.040) and Sponge-1
cells (p< 0.001) than samples S1 through S5. The standard
deviations do not differ significantly, and there were no sig-
nificant differences for the other flow cytometry parameters in
other sponge or microbial samples.
The microbial fraction samples contained ~ 10,000 times
higher counts of bacteria (HBACT) and ~ 200 times higher
counts of phytoplankton relative to the sponge cell samples
(Online Resource Table 2). Microbial cell samples contained
between 3.9 and 29.0 × 10
6
HBACT g
1
wet weight. Two
subpopulations of HBACT were found: high DNA-
containing HBACT (Bact-1) and low DNA-containing
HBACT (Bact-2) (Online Resource Figure 2). Bact-1 ranged
from 0.8 to 22.5 × 10
6
cells g
1
, and Bact-2 from 3.0 to 12.0 ×
10
6
cells g
1
.Synechococcus (SYN) abundance ranged from
0.6 to 2.4 × 10
4
cells g
1
. Phytoplankton were divided into
smaller (< 10 μm, Phyto-1) and larger (1020 μm, Phyto-2)
size fractions (Online Resource Figure 2). Phyto-1 abundance
ranged from 4.9 to 30.6 × 10
4
cells g
1
wet weight. Phyto-2
abundance ranged from 0.3 to 1.6 × 10
4
cells g
1
wet weight.
Microbial C was mostly HBACT (33% to 84%, average 69 ±
20%), with phytoplankton C ranging from 16 to 66% (average
30 ± 20%), while SYN C contributed only 0 to 1% (average
1.0 ± 0.4%) (Table 1).
C:N Molar Ratios of Whole Sponge and Isolated
Sponge and Microbial Cell Fractions
C:N molar ratios for whole sponge samples averaged 4.5 and
ranged from 4.0 to 5.4 but varied systematically with geograph-
ic location (Online Resource Table 3). Mean C:N ratio of the
isolated sponge cell fraction (6.0 ± 0.2) were significantly dif-
ferent (p= 0.016) from the mean C:N ratio of the isolated mi-
crobial cell fraction (7.3 ± 1.3, Table 2). Whole sponge samples
in the north bay had significantly higher C:N ratios than sam-
ples from mid bay (p= 0.005) and the south bay (p<0.001).
C:N ratios of whole sponge samples collected from the mid bay
did not differ from those from south bay (p=0.744). TheC:N
ratios of both the sponge cell fractions and the microbial cell
fractions were significantly higher than the C:N ratios of all
whole sponge samples from all locations (p< 0.001). The
C:N molar ratio of the plankton sample was 4.4.
Bulk δ
15
Nandδ
13
C
Bulk tissue δ
15
Nandδ
13
C values of whole sponge samples
averaged 5.1and 18.0, respectively, but varied systemat-
ically with geographic location within Kāne ohe Bay (Online
Resource Table 3). The mean bulk δ
15
N values of whole sponge
samples from the north bay were significantly higher than mid
bay (p= 0.014) and south bay (p= 0.003). However, δ
15
Nvalues
of sponges from the mid bay and south bay were not significantly
different (p= 0.798). The mean bulk δ
13
C values of sponges
from the north bay did not differ from mid bay sponges (p=
0.146), but north bay sponge mean bulk δ
13
C values were sig-
nificantly higher than south bay sponges (p< 0.001). The mean
δ
13
C values of sponges from the mid bay were also significantly
higher than the mean δ
13
C values of sponges from the south bay
(p= 0.026). The δ
15
N value of the whole plankton sample is
7.1,whereastheδ
13
Cvalueis21.8.
Aver a g e b ulk δ
15
N value of the isolated sponge cell frac-
tion was 8.1 ± 0.3and average δ
13
C value was 22.4 ±
0.4(Table 2). Average bulk δ
15
N value of the isolated mi-
crobial cell fraction was 7.9 ± 0.4and average δ
13
Cwas
26.0 ± 3.4(Table 2). The mean δ
15
N values of the sponge
cell and microbial cell fractions were not significantly differ-
ent (p= 0.200). However, the mean δ
13
C value of the sponge
cell fraction was significantly higher than the δ
13
C values of
the microbial cell fraction (p= 0.018). At all locations, the
δ
15
N value of both the sponge cell and the microbial cell
fractions were significantly higher than the δ
15
N value of
whole sponges (p< 0.001), and the δ
13
C value of both the
sponge cell and microbial cell fractions were significantly
lower than the δ
13
C values of whole sponges (p<0.001).
The δ
15
N values of whole sponge samples and sponge cell
fraction are significantly and positively correlated with C:N
molar ratios (Online Resource Figure 3). In addition, the δ
13
C
values of the microbial cell fractions were significantly and
positively correlated with C:N molar ratios (Online Resource
Figure 4).
Compound-Specific Amino Acid Analysis
Carbon isotopic composition of individual amino acids was
determined on six sponge samples with some fractions
Table 2 δ
15
N(,vs.
AIR) and δ
13
C(,vs.
V-PDB) values, as well
as carbon:nitrogen (C:N)
molar ratios, of isolated
bulk microbial and
sponge cell fractions.
M = microbial fraction;
S = sponge cell fraction
Sample δ
15
N()δ
13
C()C:N
M1 8.1 31.2 5.7
M2 8.3 23.0 8.9
M3 8.0 23.0 8.4
M4 7.8 27.2 6.9
M5 8.1 23.0 8.8
M6 7.3 28.3 6.3
M7 7.3 29.1 6.2
M8 8.0 22.8 7.5
S1 7.6 22.4 6.0
S3 8.1 22.2 6.0
S4 8.4 21.9 5.9
S5 8.3 22.4 6.1
S6 7.9 23.3 6.1
S7 7.9 22.4 5.7
S8 8.6 22.3 6.2
Shih J. L. et al.
combined in order to have sufficient material for analyses
(Table 3). Microbial cell fractions M1 and M3 as well as
sponge cell fractions S1 and S3 were combined into a single
microbial and a single sponge sample, respectively for AA-
CSIA. Three microbial cell fractions M4, M6, and M7 were
analyzed individually; however, their equivalent sponge frac-
tions had to be combined into a single sample (S4 + S6 + S7).
Although 14 amino acids were detected, the δ
13
Cvaluesof
only 12 were reliably measured in all samples (Table 3). The
δ
13
C values of amino acids ranged from 27.0 to 4.5.The
essential amino acid (Thr, Val, Leu, Phe, and Lys) average
δ
13
C value of microbial cell fraction M1 + M3 (21.1 ±
0.6) are similar and shows overlapping error with that of
sponge cell fraction S1 + S3 (21.6 ± 0.6) (Fig. 1). The
essential amino acid δ
13
Cvaluesofindividualmicrobialcell
fractions M4, M6, and M7 were not significantly different
from those of the combined sponge cell fraction S4 + S6 +
S7 (p=0.922) (Fig.1).
The carbon isotopic composition of 13 amino acids from
the plankton sample ranged from 27.8 to 11.0. The av-
erage δ
13
C value of essential amino acids in the single plank-
ton sample collected is 23.1 ± 1.1.
Recovery of sufficient material limited nitrogen isotope
analyses even more than carbon isotope analysis and only five
samples had sufficient material for analysis. In addition, only
eight amino acids were reliably measured in all samples
(Table 4). δ
15
N values ranged from 2.3 to 16.4and average
trophic amino acid (Ala, Leu, Pro, Asx, Glx)δ
15
Nvalueswere
higher (10.6 ± 3.3) than those of source amino acids (Lys,
Phe, 4.1 ± 1.3). Direct comparison of nitrogen isotopic
compositions between microbial and sponge cell fractions
could only be made for M4, M6, and M7 and S4 + S6 + S7
(Fig. 2). The source amino acid δ
15
N values of microbial cell
fractions M4, M6, and M7 were not significantly different
from those of sponge cell fraction S4 + S6 + S7 (p=0.288).
In addition, the trophic amino acid δ
15
Nvaluesofmicrobial
cell fractions M4, M6, and M7 were not significantly different
from those of sponge cell fraction S4 + S6 + S7 (p=0.267).
The δ
15
N values of 13 amino acids from the plankton sam-
ple ranged from 2.0 to 12.5. The average δ
15
Nvaluesof
source acids in the single plankton sample collected is 2.2 ±
0.7. A dendrogram produced from data clustering analysis
showed two distinct data clusters. Plankton comprised the
most distinct cluster and different from those of sponge and
microbial cell fractions, which did not separate from each
other in the cluster analysis (see Online Resource Figure 5).
Trophic position calculated from the difference in δ
15
N
values of Glx and Phe (TP
Glx-Phe
) for the combined sponge
sample (S4 + S6 + S7) is 2.1 (propagated SD = ±0.2) and the
average trophic position for samples M4, M6, and M7 is 1.9
(propagated SD = ±0.3) (Table 5). TP
Glx-Phe
of these sponge
cell and microbial cell fractions were not significantly differ-
ent (p=0.622).
Table 3 δ
13
Cvalues(, vs. V-PDB) of individual amino acids from isolated microbial and sponge cell fractions and well as plankton. M = microbial fraction; S = sponge cell fraction. Plus (+) symbols
indicate fractions combined to have sufficient material for amino acid isotope analyses. Non-essential amino acids are Ala, Gly, Ser, Pro, Asx, Glx, and Tyr. Essential amino acids are Leu, Phe, Lys, Val, and
Thr
Sample Ala Gly Ser Pro Asx Glx Tyr Leu Phe Lys Val Thr
M1 + M3 18.3 ± 0.2 11.3 ± 0.2 + 4.0 ± 1.3 19.8 ± 0.2 15.9 ± 0.1 14.6 ± 0.1 19.9 ± 0.7 26.6 ± 0.3 24.4 ± 0.1 16.2 ± 0.1 25.1 ± 0.4 13.5 ± 0.8
M4 17.5 ± 0.2 8.5 ± 0.1 0.5 ± 0.8 20.0 ± 0.2 16.8 ± 0.3 14.9 ± 0.1 24.6 ± 0.4 26.5 ± 0.1 23.5 ± 0.2 17.6 ± 0.6 24.5 ± 0.1 15.3 ± 0.4
M6 17.2 ± 0.2 7.0 ± 0.7 2.9 ± 0.8 20.2 ± 0.2 17.2 ± 0.3 15.4 ± 0.2 25.2 ± 0.8 25.7 ± 0.1 23.4 ± 0.3 17.4 ± 0.4 24.8 ± 0.4 15.6 ± 0.4
M7 21.0 ± 0.2 9.4 ± 0.9 0.5 ± 0.9 19.9 ± 0.2 16.6 ± 0.1 15.9 ± 0.8 22.6 ± 0.3 26.5 ± 0.5 24.4 ± 0.3 18.5 ± 0.3 24.1 ± 0.3 15.2 ± 0.1
S1 + S3 19.0 ± 0.4 12.4 ± 0.5 17.3 ± 0.1 19.5 ± 0.3 19.1 ± 0.1 15.8 ± 0.1 26.3 ± 0.4 27.0 ± 0.1 24.2 ± 0.1 18.2 ± 0.3 23.9 ± 0.2 14.9 ± 0.2
S4 + S6 + S7 17.3 ± 0.3 13.4 ± 0.3 + 4.5 ± 1.2 19.6 ± 0.4 17.2 ± 0.2 14.6 ± 0.3 23.3 ± 0.2 26.3 ± 0.1 23.8 ± 0.1 17.9 ± 1.0 25.3 ± 0.9 13.8 ± 0.5
Plankton 20.8 ± 0.3 19.5 ± 0.3 11.0 ± 0.6 17.3 ± 0.4 15.4 ± 0.2 16.1 ± 0.4 25.2 ± 0.7 27.8 ± 0.2 24.9 ± 0.4 19.3 ± 1.0 27.2 ± 0.1 17.9 ± 0.4
Trophic Ecology of the Tropical Pacific Sponge Mycale grandis Inferred from Amino Acid Compound-Specific...
The difference in the average δ
15
Nvaluesoftrophicand
source amino acids, a proxy measure of trophic position, of
the sponge cell fraction (sample S4 + S6 + S7) was 6.9 ±
1.3and that of the microbial cell fractions (samples M4,
M6, and M7) was 5.9 ± 1.2(Table 5). These proxy mea-
surements of trophic position of the microbial and sponge cell
fractions were not significantly different (p= 0.540). In addi-
tion, the difference in the weighted mean δ
15
Nvaluesoftro-
phic and source amino acids of the sponge cell fraction (sam-
ple S4 + S6 + S7) was 9.4 (propagated SD = 0.4) and 8.9
(propagated SD = 0.3) for the microbial cellfractions (samples
M4, M6, and M7). This trophic proxy of the microbial and
sponge cell fractions was also not significantly different (p=
0.286).
Summed Variance in δ
15
N Values of Trophic Amino
Acids (ΣV)
V values of cell fractions ranged from 2.0 to 3.0 (Table 6). V
values of microbial cell fractions M4, M6, and M7 were not
significantly different from those of sponge cell fraction S4 +
S6 + S7 (p=0.230).
Discussion
Sponge-associated microbial symbionts play an important
role in the trophic ecology of sponges. While the sponge
holobiont has been shown to be capable of DOM uptake and
ultimately the incorporation of both carbon and nitrogen, the
exact mechanisms and the role(s) microbes may play in this
process has not been fully described. Separate analysis of
sponge and symbiotic microbial cells here reveal details about
the relationship and mechanism of nutrient transfer from sym-
biont microbes to the sponge host. The separated sponge cell
and microbial cell fractions contained distinct cell composi-
tions, with the sponge fraction dominated by large (> 5 μm
diameter) sponge cells and the microbial cell fraction predom-
inantly composed of heterotrophic bacteria. Despite large and
systematic variation of δ
15
Nandδ
13
C values in whole sponge
samples throughout Kāne ohe Bay, the δ
13
C values of the
essential amino acids and δ
15
N values of the source amino
acids were not statistically different between the two cell frac-
tions. The indistinguishable amino acid isotopic compositions
and high ΣV values suggest that M.grandis, despite having
chlorophyll-containing cells within some sponge cells, pri-
marily acquires C and N through direct transfer of bacterially
Table 4 δ
15
N values (vs. AIR) of individual amino acids from
isolated microbial and sponge cell fractions as well as plankton. M =
microbial fraction; S= sponge cell fraction. Plus (+) symbols indicate
fractions combined to have sufficient material for amino acid isotope
analyses. Trophic amino acids are Ala, Glx, Leu, Pro, and Asx. Source
amino acids are Phe, Tyr, and Lys. ND indicates no data
Sample Ala Glx Leu Pro Asx Phe Tyr Lys
M1 + M3 5.9 ± 0.1 16.1 ± 0.1 10.9 ± 0.4 9.9 ± 0.3 13.7 ± 0.2 2.7 ±0.2 2.5 ± 0.5 4.4 ± 0.4
M4 5.7 ± 0.2 15.2 ± 0.0 9.8 ± 0.1 8.6 ± 0.4 11.8 ± 0.1 5.1 ± 0.2 4.2 ± 1.3 3.4 ± 0.2
M6 6.5 ± 1.0 14.3 ± 0.4 9.6 ± 0.7 9.3 ± 1.1 10.9 ± 0.2 6.5 ± 0.5 ND 3.6 ± 0.3
M7 ND 14.0 ± 0.3 9.1 ± 0.5 7.8 ± 0.6 10.8± 0.4 2.3 ± 0.5 1.1± 0.3 3.4 ± 0.5
S4 + S6 + S7 6.4 ± 0.2 16.4 ± 0.1 12.2 ± 0.3 9.7 ± 1.1 14.1 ± 0.2 4.6 ± 0.3 2.4 ± 0.3 5.2 ± 0.7
Plankton 12.5 ± 0.2 11.6 ± 0.2 6.7 ± 0.4 12.0 ± 0.3 8.9 ± 0.4 0.6 ± 0.4 4.6 ± 0.7 3.8 ± 0.5
15
N, ‰
0
2
4
6
8
10
12
14
16
Microbial Cells
Sponge Cells
Plankton
Ala
Glx
Leu
Pro
Asx
Phe
Lys
Trophic AA Source AA
Fig. 2 Trophic and source amino acid δ
15
Nvalues(vs. AIR) in
microbial and sponge cell fractions isolated from Mycale grandis and
from a plankton tow collected in south Kāne ohe Bay
13
C, ‰
-28
-26
-24
-22
-20
-18
-16
-14 Microbial Cells
Sponge Cells
Plankton
Leu
Phe
Lys
Val
Thr
Fig. 1 Essential amino acid δ
13
Cvalues(, vs. V-PDB) in microbial and
sponge cell fractions isolated from Mycale grandis and from a plankton
tow collected in south Kāne ohe Bay
Shih J. L. et al.
synthesized amino acids, and not from feeding on planktonic
microbes or detrital components from the water column.
Therefore, microbial symbionts appear to play a major role
in the acquisition of C and N from DOM uptake and its sub-
sequent transfer and utilization by M.grandis.
Separation of Sponge and Microbial Cell Fractions
Determining the distinct isotopic composition of the sponge
and its associated microbes required separation of these two
fractions. Flow cytometry revealed fractions distinguished by
size (light scatter), pigment (chlorophyll and phycoerythin
fluorescence), and DNA content. Epi-fluorescence microsco-
py showed large (~ 5 μm nuclei) cells within the sponge tissue
samples that were largely devoid of fluorescence and hence
composed of only sponge cells. Consequently, these results
indicate that the sponge fractions contained predominantly
sponge cells and that the microbial fractions contained pre-
dominantly smaller prokaryotic cells.
Bulk δ
15
Nandδ
13
C values in the whole sponge, sponge,
and microbial cell fractions also suggest good separation of
the cell fractions (Fig. 3). Although there is overlap in δ
13
C
and δ
15
N values between some sponge and microbial cell
fractions, the average difference between these two fractions
is quite large (3.5) and the mean C:N ratio of sponge cell
and microbial cell fractions are statistically different.
A weak correlation exists between the δ
13
Cvaluesofbulk
microbial cells and the C contribution of bacteria to the mi-
crobial cell fraction (Online Resource Figure 6). A higher C
contribution from bacterial cells in this fraction results in low-
er δ
13
C values. In addition, a plot of δ
13
C values versus the
inverse of the bacterial biomass (1/HBACT) yields a y-
intercept close to 29, which closely resembles samples
M1, M4, M6, and M7. Some microbial cell fractions (M2,
M3, M5, and M8) were also found to have unexpectedly high
C:N ratios and the δ
13
C values of the microbial cell fractions
are significantly and positively correlated with C:N ratios
(Online Resource Figure 4), suggesting some microbial cell
fractions may have contained carbonate that would increase
both C:N ratios and δ
13
Cvalues.
Based on these relationships, we suggest that microbial cell
fractions with higher C:N values are not representative of the
pure microbial fraction, while the lower C:N values found in
samples M1, M4, M6, and M7 are more representative and
distinctly different from the composition of sponge cell
fractions.
Isolated sponge and microbial cell fractions differ signifi-
cantly from whole sponge tissue samples in C:N, δ
15
N, and
δ
13
C values. In the Demospongiae, the internal sponge matrix
is dominated by gelatinous collagen-rich mesohyl reinforced
by a dense network of fibrous spongin that provides structure
and flexibility. Spongin is a modified collagen protein secreted
by collagen-producing sponge cells within the mesohyl, and
together spongin and mesohyl constitute the majority of bulk
sponge tissue. Diet-to-collagen isotope fractionation in bone
collagen is known to resultin
13
Cenrichmentinbonecollagen
of about 5relative to other biomolecules from plants and
13C, ‰
-32 -30 -28 -26 -24 -22 -20 -18 -16 -14
15N, ‰
0
2
4
6
8
10
Bulk Sponge
Isolated Sponge Cells
Isolated Microbial Cells
Fig. 3 Bulk δ
13
C(, vs. V-PDB) and δ
15
Nvalues(vs. AIR) of whole
sponges and the sponge and microbial cell isolates. The whole sponge
carbon and nitrogen isotopic composition of the sponges that the cells
were isolated from was not measured. Note that the sum of isolated
sponge cell + microbial cell isolates whole sponge, since whole
sponge samples include structural material not present in either fraction
Table 5 Mycale grandis isolated sponge (S) and microbial (M) cell
fractions trophic position (TP
Glx-Phe
) and trophic position proxies (TPP).
TPP calculated using average of δ
15
N values of trophic and source amino
acids (average), and TPP calculated using weighted mean δ
15
Nvaluesof
trophic and source amino acids (weighted), along with propagated stan-
dard deviations
Sample TP
Glx-Phe
TPP (average) TPP (weighted)
S4 + S6 + S7 2.1 ± 0.2 6.9 ± 1.3 9.4 ± 0.3
M4 1.9 ± 0.2 6.0 ± 1.0 11.1 ± 0.2
M6 1.6 ± 0.2 5.1 ± 1.4 7.9 ± 0.4
M7 2.1 ± 0.2 6.6 ± 1.1 7.7 ± 0.4
M4 + M6 + M7 (average) 1.9 ± 0.3 5.9 ± 1.2 8.9 ± 0.3
Table 6 Summed variance in the δ
15
N values of select trophic amino
acids (ΣV) of isolated sponge (S) cell and microbial (M) cell fractions of
Mycale grandis
Sample Summed variance (ΣV)
S4 + S6 + S7 3.0
M1 + M3 2.9
M4 2.6
M6 2.0
M7 2.4
Average microbial (all) 2.5
Average microbial (M4, M6, M7) 2.3
Trophic Ecology of the Tropical Pacific Sponge Mycale grandis Inferred from Amino Acid Compound-Specific...
animals (e.g., see Fernandes et al. [61] and references therein).
We suggest that similar carbon isotope fractionation can ex-
plain the ~ 5difference between the spongin-rich whole
sponge and sponge cell fraction δ
13
Cvalues.
The C:N ratio of the sponge cell fraction (average of S4 +
S6 + S7) was significantly lower relative the C:N of the mi-
crobial fraction (average of M4 + M6 + M7), indicating fun-
damental differences in the composition of the two fractions.
In addition, the microbial cell fractions also exhibited lower
average bulk δ
13
C values compared to the bulk δ
13
Cvaluesof
sponge cells. Higher lipid-to-biomass content drives δ
13
C
values lower and increases C:N ratio [62,63]. Thus, higher
lipid content in microbial cells may contribute to observed
δ
13
C values and C:N ratios. While the C:N ratio and the rel-
ative carbohydrate-lipid-protein composition of sponge-
associated bacteria are not known, the sponge cell fraction
may have a higher protein and carbohydrate composition rel-
ative to lipid than the microbial samples, which may be
reflected in the observed C:N ratios. Aside from lipid content
effects on C:N and δ
13
C differences, the protein fraction con-
tains dietary information about the transfer of amino acids,
and the fractionation between trophic and source amino acids
in N and non-essential and essential amino acids in C.
The δ
15
N values of the sponge cell fraction were signifi-
cantly different from those of whole sponge (Online Resource
Figure 3); however, this difference was likely derived from the
varying N isotopic composition of nutrients in different re-
gions of Kāne ohe Bay. Nitrogen inputs vary with seasonal
patterns and weather (e.g., heavy rainfall and wind). Stream
runoff and anthropogenic inputs from urban development,
such as coastal residential housing, Heeia Harbor, and com-
mercial properties are also sources of N isotope variability
across the bay [64,65]. The higher δ
15
Nvaluesmeasuredin
samples from the north bay may indicate input from more
numerous residential cesspools in the watershed adjacent to
the northern portion of the bay [66], as NO
3
associated with
sewage input is typically enriched in
15
Nrelativetothatin
seawater [67,68]. Inputs of submarine groundwater discharge
[69] would also result in higher δ
15
Nvalues[70,71].
Amino Acid Isotopic Compositions of Sponge Cell
Fractions and Plankton
Although the flow cytometry, microscopy, C:N ratios, and
carbon isotopic compositions of microbial and sponge cell
fractions were distinctly different, the carbon and nitrogen
isotopic compositions of amino acids in both fractions were
remarkably similar (Tables 3and 4,Figs.1and 2). This sim-
ilarity in bacterial and sponge cell amino acid δ
13
Candδ
15
N
values strongly suggests that M.grandis is acquiring amino
acids directly from its heterotrophic bacteria and not through
feeding on Synechococcus spp.or other planktonic photoau-
totrophs. This is because it is unlikely that Synechococcus or
other autotrophs would synthesize amino acids with C and N
isotopic compositions identical to the pattern found in the
heterotrophic microbial cells that dominate the microbial cell
fractions.
Indo-Pacific sponges have long been considered to be
phototrophic [25,72], and M.grandis hosts cyanobacterial
phylotypes [73]. Among cyanobacteria, Synechococcus and
Prochlorococcus are the dominant groups in most oceans
and have been found in at least 26 Demospongiae families
[16]. A monophyletic Synechococcus spongiarumclade
that is phylogenetically distinct from the nearest free-living
Synechococcus relative has been reported to be present in 18
sponge species from various geographic locations and is the
most prevalent photosynthetic symbiont present in marine
sponges [74,75]. Although cyanobacterial symbionts can
comprise 25 to 50% of a spongescellularvolume[76], rela-
tively little is known about the metabolic exchange and eco-
logical interactions between sponges and their microbial
symbionts.
Synechococcus is the dominant cyanobacteria taxon in
Kāne ohe Bay and contributes 35% of phytoplankton bio-
mass, and is the main autotroph in the microbial to metazoan
consumer food web, particularly during dry conditions (i.e.,
low stream flow) [77]. Consequently, the δ
13
Cvaluesofes-
sential amino acids and the δ
15
N values of source amino acids
in the plankton net sample (63250 μm) should reflect those
of Synechococcus. Indeed, these average values, 23.4 ±
1.1δ
13
C and 2.2 ± 0.7δ
15
N, were lower than the essen-
tial amino acids and source amino acids found in the sponge
cell fraction (21.4 ± 1.5δ
13
C and of 4.9 ± 0.7δ
15
N,
Figs. 1and 2).
Sponge cells with high DNA, chlorophyll, and phycoery-
thrin signals represented ~ 1/3 of the sponge cell biomass and
were likely sponge cells with phytoplankton prey within them.
Many Synechococcus species contain phycoerythrin, and
some sponges have been shown to prey on Synechococcus
in feeding clearance studies [7880]. It is unknown whether
M.grandis consumes Synechococcus prey directly and we did
not perform Synechococcus feeding experiments. However, in
our M.grandis samples, Synechococcus represented < 1% of
the carbon contribution in the microbial cell fraction, even
though microscopy showed that Synechococcus was the most
abundant sponge-associated microbe in M.grandis.Although
Synechococcus are ~ 2-3Xs larger than most bacterial cells
and some may have been lost during cell fraction separation,
their low abundance and differing isotopic signals suggest
their negligible importance in the acquisition of amino acids
by M.grandis sponge cells.
Lastly, using the δ
13
C values of essential amino acids and
the δ
15
N values of source amino acids, the net plankton sam-
ple forms a distinct end-member that clusters separately from
the microbial and sponge cell fractions (Online Resource
Figure 5). This distinct clustering of amino acid δ
13
C and
Shih J. L. et al.
δ
15
N values provides support for microbial symbionts as be-
ing the source for amino acids in the sponge M.grandis,as
opposed to plankton-derived nutrition or DOM uptake. Our
results however cannot distinguish between the possibility
that the essential and source amino acids in M.grandis sponge
cells fraction were acquired from planktonic heterotrophic
bacteria. However, if planktonic heterotrophic bacteria were
the source of these amino acids in sponge cells, it would re-
quire that they took up DOM and synthesized essential amino
acid and source amino acids with δ
13
Candδ
15
Nvaluessig-
nificantly different from that we measured in our plankton
sample but identical to that in the microbial and sponge cells
isolated from M.grandis. Given the similarity in isotopic
compositions of essential and source amino acids in the mi-
crobial and sponge cell fractions isolated from M.grandis,the
most parsimonious explanation is that microbial symbionts
hosted by the sponge were responsible for uptake of DOM
and the synthesis of amino acids that were translocated to
sponge cells.
Summed Variance in δ
15
N Values of Trophic Amino
Acids (ΣV)
ΣV is a proxy for heterotrophic bacterial re-synthesis of or-
ganic matter, as partial re-synthesis introduces isotopic vari-
ability among trophic amino acids in bacterial cells [40,41,
81]. Heterotrophic reworking of proteinaceous material occurs
through a range of processes, including extracellular hydroly-
sis [42], de novo synthesis and selected re-synthesis, salvage
pathways incorporating amino acids into new biomass, and
strict catabolism [40]. The ΣV index is a measure of the rela-
tive variability in the δ
15
Nvaluesofselecttrophicaminoacids
due to microbial alteration [82]. Patterns in ΣV can differen-
tiate microbial processing from eukaryotic synthesis of amino
acids. Values above ΣV ~ 2 are generally considered a thresh-
old for organic matter resulting predominantly from bacterial
re-synthesis of detrital materials, although consumption of
particles by zooplankton can also result in a slight increase
in ΣVvalues[40].
The V value of the net plankton sample was 2.0, whereas
V values in sponge and microbial fractions tended to be
higher (2.03.0) (Table 6) and were consistent with heterotro-
phic microbial alteration of amino acids. The V values in
microbial and sponge cell fractions were similar to those
found in cultures of heterotrophic bacteria and microbially
reworked particles in the ocean [4042] and were higher than
values found in phytoplankton and zooplankton [40].
McCarthy et al. [40] observed typical values of ΣVrange from
~ 0 to 1 for phytoplankton, 1 to 1.5 for zooplankton, and up to
~ 3 for detrital POM in surface sediment traps along the equa-
torial Pacific. Yamaguchi and McCarthy [82]alsomeasured
high values of ΣV (~ 2 to 4) in high molecular weight dis-
solved organic matter in the Pacific Ocean north of Hawaii.
These researchers also showed that ΣV values increased from
surface waters (21 m, ΣV = ~ 2) to mesopelagic depths (ΣV=
~ 2.8 at 915 m and ~ 4 at 670 m). In contrast, they found
500 kDa to ~ 1 μm particles from 25 to 750 m had ΣVvalues
less than ~ 2. Consequently, Yamaguchi and McCarthy [82]
suggested that high ΣV values are found in DOM that has
been bacterially altered and in bacterial cells which have part-
ly re-synthesized amino acids.
The ΣV of the microbial and sponge cell fractions can
reveal the origin of amino acids. The high values observed
in the sponge cell fractions (S4 + S6 + S7) suggest that amino
acids were not acquired directly from the consumption of
fresh phytoplankton. Although higher ΣV values were ob-
served in zooplankton relative to phytoplankton in the equa-
torial Pacific Ocean [40], those changes were small (increase
from ~ 0.7 to 1.5). Sponges have the capacity to incorporate
DOM [7,8,12,26]. Thus, the high ΣVvaluesobservedin
microbial and sponge cell fractions may be evidence of uptake
of DOM with high ΣV values. If bacteria in M.grandis play a
significant role in DOM uptake, these microbes could acquire
at least some of their high ΣVvalues from DOM and transfer
those partially re-synthesized amino acids to the host sponge
cells. However, the lack of statistical difference between the
ΣV values of sponge cell and microbial cell fractions supports
the conclusion that the high ΣV values observed in the sponge
cell fraction were inherited from their microbial symbionts,
rather than the sponge cells directly taking up DOM from
the water column.
Trophic Position and Trophic Proxy
No significant differences were observed in trophic position
determined from AA-CSIA between the sponge cell and mi-
crobial cell fractions (Table 5). Although calculation of the
trophic position from AA-CSIA has become common in
metazoan food webs, questions remain about the accuracy of
βand Δvalues [34,57]. In addition, little is known about these
values in microbial food webs [83]. Assuming that βand Δ
values used in metazoan food webs apply, the average trophic
position calculated for the microbial cell fraction (1.9 ± 0.3) is
not different from that determined for the sponge fraction an-
alyzed (2.0 ± 0.2). Assuming the βand Δvalues remain rea-
sonably constant, calculation of trophic position from AA-
CSIA is highly dependent upon the difference between the
δ
15
N values of trophic and source amino acids.
Trophic position proxy (TPP) removes the reliance on as-
sumed knowledge of βand Δvalues, and is therefore based
only on the difference in δ
15
Nvaluesoftrophicandsource
amino acids [57,84]. Metazoan consumption results in
15
N
enrichment of trophic amino acids relative to source amino
acids [3436]. TPP values for sponge cells (S4 + S6 + S7)
were not greater than the TPP values of the complementary
microbial cell samples (M4, M6, and M7) when determined
Trophic Ecology of the Tropical Pacific Sponge Mycale grandis Inferred from Amino Acid Compound-Specific...
from average δ
15
N values of amino acids (Table 5). TPP using
weighted δ
15
N averages based on the uncertainty of the mea-
surements also revealed no significant difference in TPP be-
tween the sponge and its associated microbes (Table 5).
Phagotrophic protists do not necessarily exhibit the system-
atic
15
N trophic enrichment that is well established for meta-
zoan consumers. Salvage pathway incorporation of amino
acids was thought to be restricted to bacteria, but the δ
15
N
values of trophic and source amino acids in some protistan
consumers are not significantly different from those in their
prey [85], with the exception of Ala [85,86]. While a similar
effect cannot currently be eliminated as a possibility for the
lack of difference observed between isotopic patterns of ami-
no acids in sponge and microbial cell fractions, all other evi-
dence indicates that direct transfer and incorporation of amino
acids from microbes to sponge is more likely.
First, the δ
15
N values of source amino acids and the δ
13
C
values of essential amino acids are not statistically different in
microbial and sponge cell fractions, suggesting that amino acids
in the sponge fraction are directly transferred from the sponge-
associated microbes to sponge cells without isotope fraction-
ation. Unfortunately, no C isotope results are available from the
experiments of Gutiérrez-Rodríguez et al. [85] or Décima et al.
[86] to further corroborate these results. Second, sponge and
microbial cell fractions ΣV values are not statistically different
and the values are indicative of heterotrophic microbial alter-
ation of amino acids (Table 6). ΣVvaluesofalgaDunaliella
isolated from the first stage of the chemostat experiments of
Gutiérrez-Rodríguez et al. [85] averaged 3.1, whereas ΣV
values of protists in stages 2 (light) and 3 (dark) were 1.4. It
is unknown why the Dunaliella ΣV values in these experiments
are so high. Unfortunately, these data are too few however to
draw conclusion about whether sponge cells feeding
phagotrophically on their host microbial cells are responsible
for the similarity in trophic position between these cell types as
has been observed elsewhere in protists [10,87].
Should sponges behave as typical metazoans and consume,
digest, and assimilate bacterial cells, they would exhibit a dif-
ference in trophic position through obligate deamination and
transamination in amino acids derived from their bacterial prey.
Instead, it appears that amino acids are transferred from hetero-
trophic bacteria to the host by translocation in a manner similar
to those found in scleractinian corals and its endosymbionts
(Family Symbiodiniaceae) in the transfer of nutrients that sup-
port growth and metabolism [88,89]. It is also possible that the
transfer of nutrients is from the phagocytosed microbes they
host. Corals harbor a variety of symbiotic archaea and bacteria
[90], and the translocation of C-rich photosynthates to the host
may help the symbionts maintain a favorable C:N ratio [91].
While corals are photoautotrophic, they also rely on prey cap-
ture and particle feeding to meet their N requirements. Sponges
may likewise be able to undergo shifts between translocation-
like nutrient acquisition and heterotrophic feeding as observed
in coral [92] under different environmental conditions. The
amino acid isotopic compositions of sponge and microbial cells
suggest that M. grandis acquires C and N predominantly
through translocation of amino acids or through some kind of
direct assimilation of their host microbes. It is not currently
known if translocation of amino acids can occur in sponges;
however, our results are consistent with the occurrence of the
translocation of products between sponge cells we analyzed and
symbiotic microbes that would not produce isotopic fraction-
ation through the severing of amine bonds.
Implications for Amino Acid Metabolism in Sponges
The lack of
15
N enrichment in trophic amino acids relative to
source amino acids in sponges is surprising and may have
implications for amino acid metabolism in sponges. The ab-
sence of change in the δ
15
N value of Glx between bacterial
and sponge cells is particularly surprising since glutamate is a
key intermediate in the Krebs cycle and plays a fundamental
role in amino acid biosynthesis in metazoans that link protein
and energy metabolism [93]. However, as mentioned above,
phagotrophic protists do not necessarily exhibit the systematic
15
N trophic enrichment that is seen in metazoan consumers
and choanocytes phagocytized a significant proportion of the
small particles they capture [94]. Early studies [95]showed
that phagosomes containing particles were quickly transferred
from choanocytes to archaeocytes in the mesophyll where
digestion and assimilation continues. Sponges can recognize
and discriminate microbes using immune signaling [18]and
Leys et al. [10] showed microscopic evidence for large num-
bers of symbiont microbes being phagocytosed in archaeocyte
cells. Although choanocyte and archaeocyte phagocytose, di-
gest, and assimilate prey, excretion of metabolic products oc-
curs primarily in spherulous and granular cells in the sponge
mesophyll [96]. Since primary nitrogen isotope effects can
only occur when nitrogen bonds are broken or formed,
15
N
enrichment in trophic amino acids relative to source amino
acids might be restricted to spherulous and granular cells in
the sponge mesophyll where amino acid transamination and
deamination occurs. With regard to our results, spherulous and
granular sponge cells may not have been in high concentration
in our sponge cell fraction and therefore we did not observe
15
N enrichment in trophic amino acids relative to source ami-
no acids typical of metazoans.
In summary, the lack of
15
N enrichment in trophic amino
acids relative to source amino acids in sponge cells we ana-
lyzed may be expected. The separation scheme we used con-
centrated choanocyte cells. The similarity in bacterial and
sponge cell δ
13
C values of essential amino acids and δ
15
N
values of source amino acids strongly suggests that M.grandis
is acquiring amino acids directly from its heterotrophic bacte-
ria. The isotopic composition of these amino acids in choano-
cyte cells may have been acquired through translocation of
Shih J. L. et al.
bacterial amino acids or through bacterial cell phagocytosis. It
is possible that amino acid and energy metabolism may occur
in spherulous and granular cells not measured in this study.
This hypothesis could be tested by analyzing the isotopic
composition of spherulous and granular cells or perhaps spon-
gin collagen fibers secreted by spongocyte cells.
Conclusions
Evidence of symbiont-derived nutrition has been studied in
phototrophic sponges [44,97] and the incorporation of algal
and coral-derived DOM into sponge tissue [5,8,12,26]. In
both cases, evidence suggests that the microbial symbionts
provide nutrition to the host sponge. While primary producers
have been identified as the original source of C and N to reefs
and the sponge holobiont, previous experiments did not di-
rectly demonstrate that C and N were transferred as amino
acids from their symbiotic microbes directly to sponge cells.
Here, we propose that bacterial symbionts in M.grandis pro-
vide an intermediate step in the transfer of C and N from DOM
as suggested by others [912]. It is unlikely that identical
isotopic compositions of amino acids in sponge and microbial
cells would occur from direct transfer of amino acids from
planktonic heterotrophic bacteria to sponge cell, as microbial
symbionts of M.grandis and planktonic heterotrophic bacteria
would be expected to have different amino acid isotopic com-
positions. Rather, we suggest that the heterotrophic bacteria
hosted by M.grandis are consuming DOM, re-synthesizing
the organic material, and passing on nutrition to the sponge as
amino acids through translocation. This scenario is consistent
with no change in trophic position and absence of amino acid
isotope fractionation between the microbe and the host sponge
cells analyzed, as it implies that no amine bonds were broken
in this transfer and that is inclusive of essential and trophic
and sourceamino acids. Thus, as previously recognized,
nitrogen cycling within marine sponges is likely more com-
plex than previously assumed.
Acknowledgments We thank Dr. Laura Núñez-Pons (Stazione Zoologica
Anton Dohrn) for guidance and assistance in the field, Dr. Ruth Gates
(Hawai i Institute of Marine Biology) for invaluable input and use of
laboratory facilities, Leon Weaver (Hawai i Institute of Marine Biology)
for assistance in the maintenance of experimental facilities, and Jen
Davidson (Hawai i Institute of Marine Biology) for technical assistance
in the laboratory. This is Hawai i Institute of Marine Biology contribution
number 1764and the School of Ocean andEarth Science and Technology
contribution number 10737.
Funding This study was funded by a grant/cooperative agreement from
the National Oceanic and Atmospheric Administration, Project #R/TR-
18, which is sponsored by the University of Hawaii Sea Grant Collect
Program, SOEST, under Institutional Grant No. #NA14OAR4170071
from NOAA Office of Sea Grant, Department of Commerce. The views
expressed herein are those of the authors and do not necessarily reflect the
views of NOAA or any of its subagencies.
Compliance with Ethical Standards
Conflict of Interest The authors declare that they have no conflict of
interest.
Ethical Approval This article does not contain any studies with human
participants or animals performed by any of the authors.
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Shih J. L. et al.
... Sponges also consume DOM, and the microbiome of high microbial abundance sponges (HMA) can consume DOM and translocate byproducts of their metabolism (e.g., dissolved free amino acids) to the host (de Goeij et al. 2008, Freeman and Thacker 2011, Maldonado et al. 2012, Thacker and Freeman 2012, Fiore et al. 2013, 2020, de Goeij et al. 2013, 2017, Shih et al. 2020. However, many of these studies quantified dissolved organic carbon (DOC) but not dissolved organic nitrogen (DON), and while DON is generally an order of magnitude lower in terms of availability compared with DOC , it should be quantified to obtain a complete representation of sponge DOM consumption and reprocessing. ...
... The results of our feeding measurements confirm that A. tubulata, and its microbiome, also utilize significant quantities of DOM in their diets. While it has been shown that sponge cells can utilize DOM (Achlatis et al. 2019), the experimental evidence also shows that the primary consumers of DOM within the sponge, particularly algal derived DOM, are the prokaryotic microbiome (Zhang et al. 2019, Rix et al. 2020 which can then transform and translocate DOM to the host (Shih et al. 2020). The microbiome of A. tubulata, particularly the high proportion of Chloroflexi bacteria, has also been shown to have broad metabolic potential for the use of labile, semi-labile, and refractory DOM. ...
... The microbiome of A. tubulata, particularly the high proportion of Chloroflexi bacteria, has also been shown to have broad metabolic potential for the use of labile, semi-labile, and refractory DOM. However, it is unknown what proportion of this DOM is provided to A. tubulata from its microbiome in the form of translocated products or via the phagocytosis of symbionts (Leys et al. 2018, Shih et al. 2020. The proportional uptake of POC to DOC (2-4% and 96-98%, respectively) found in this experiment was similar to values on emergent HMA sponges on coral reefs , Wooster et al. 2019), but the POC proportion was lower than observed in other studies (McMurray et al. 2018, Rix et al. 2020. ...
Article
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On Caribbean coral reefs, sponges are important members of the benthic community and play multiple roles in ecosystem structure and function. They have an important role in benthic‐pelagic coupling, consuming particulate organic matter (POM) and dissolved organic matter (DOM) and in turn providing food in the form of sponge biomass or the release of detritus for a variety of coral reef organisms. Throughout the Caribbean, sponges show consistent increases in their abundance and growth rates as depth increases into the mesophotic zone (30–150 m). This has been hypothesized to be driven by bottom‐up forces, particularly the increased supply of nitrogen‐rich POM in mesophotic coral reef ecosystems (MCEs). Here, we tested the hypothesis that the sponge, Agelas tubulata, exhibits increased growth rates on MCEs relative to shallow reefs on Grand Cayman Island and that this is driven by bottom‐up forcing. We observed increased growth rates in mesophotic A. tubulata, compared with shallow conspecifics, despite variability in feeding on both POM and DOM. Mesophotic sponges, however, were consistently exposed to greater amounts of POM, which was seasonally variable unlike DOM. Changes in stable isotopic signatures, and higher feeding rates with increasing depth, were consistent with increasing rates of growth in sponges as depth increases. These observations support the hypothesis that mesophotic sponges have higher growth rates due to increased POM availability and consumption over time. The results of this study illustrate the crucial role that bottom‐up forcing has in the structuring of sponge communities on both shallow and mesophotic Caribbean coral reefs and the importance of POM as a source of nitrogen in sponge diets.
... Isotopic analysis of amino acids in all samples (coral host, symbiont algae, plankton) was performed by subjecting tissue samples to acid hydrolysis, carboxyl terminus esterification, and amine group trifluoroacetylation (Hannides et al. 2013;Shih et al. 2020) (see Supporting Information Appendix S1). Acid hydrolysis was performed by heating (150 C) approximately 15 mg of tissue in 6 N HCl, evaporating to dryness, redissolving hydrolysate in 0.01 N HCl, filtering (0.2-μm polyethersulfone filter), purifying by cation exchange (Dowex 50WX8-400), and amino acid elution with ammonium hydroxide. ...
... Hydrolyzed tissues were esterified and the amine group was trifluoroacetylated. Finally, solvent extraction in Pbuffer (KH 2 PO 4 + Na 2 HPO 4 in milli-Q water, pH 7) was used to further purify samples (Shih et al. 2020). Chloroform was used to partition acylated amino acids, and following solvent evaporation sample trifluoracetylation was repeated to maximize derivitization. ...
... where δ 15 N x is the nitrogen isotopic value of a specified amino acid within the trophic or source category, and σ x is the standard deviation of the amino acid based on triplicate analysis (Shih et al. 2020). ...
Article
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Compound‐specific isotope analyses (CSIA) and multivariate “isotope fingerprinting” track biosynthetic sources and reveal trophic interactions in food webs. However, CSIA have not been widely applied in the study of marine symbioses. Here, we exposed a reef coral (Montipora capitata) in symbiosis with Symbiodiniaceae algae to experimental treatments (autotrophy, mixotrophy, heterotrophy) to test for trophic shifts and amino acid (AA) sources using paired bulk (δ13C, δ15N) and AA‐CSIA (δ13CAA, δ15NAA). Treatments did not influence carbon or nitrogen trophic proxies, thereby not supporting nutritional plasticity. Instead, hosts and symbionts consistently overlapped in essential‐ and nonessential‐δ13CAA (11 of 13 amino acids) and trophic‐ and source‐δ15NAA values (9 of 13 amino acids). Host and symbiont trophic‐δ15NAA values positively correlated with a plankton end‐member, indicative of trophic connections and dietary sources for trophic‐AA nitrogen. However, mass balance of AA‐trophic positions (TPGlx–Phe) revealed heterotrophic influences to be highly variable (1–41% heterotrophy). Linear discriminant analysis using M. capitata mean‐normalized essential‐δ13CAA with previously published values (Pocillopora meandrina) showed similar nutrition isotope fingerprints (Symbiodiniaceae vs. plankton) but revealed species‐specific trophic strategies. Montipora capitata and Symbiodiniaceae shared identical AA‐fingerprints, whereas P. meandrina was assigned to either symbiont or plankton nutrition. Thus, M. capitata was 100% reliant on symbionts for essential‐δ13CAA and demonstrated autotrophic fidelity and contrasts with trophic plasticity reported in P. meandrina. While M. capitata AA may originate from host and/or symbiont biosynthesis, AA carbon is Symbiodiniaceae‐derived. Together, AA‐CSIA/isotope fingerprinting advances the study of coral trophic plasticity and are powerful tools in the study of marine symbioses.
... Species with dense, collagenous tissue and those containing symbionts and host cells with similar or overlapping sizes pose particular challenges and would require additional optimization steps. Nevertheless, this technique has been successfully used in a range of sponge species to infer trophic relationships between sponges and symbionts using natural stable-isotopic signatures of separated fractions (Freeman & Thacker 2011, Shih et al. 2020, and to follow the transfer of symbiontderived inorganic C and N to host cells (Fiore et al. 2013, Freeman et al. 2013. ...
... The molar C:N ratios of our sponge cell and microbial fractions were significantly different, indicating good separation of the fractions (Fiore et al. 2013, Shih et al. 2020, but a degree of cross-contamination does occur. However, similar values for host/symbiont contributions to DOM uptake were found in D. avara and Aplysina aerophoba using cell-separation and NanoSIMS (Rix et al. 2020), confirming the validity of the cell-separation technique. ...
Article
Full-text available
Sponge-microbe symbioses underpin the ecological success of sponges in many aquatic benthic ecosystems worldwide. These symbioses are often described as mutually beneficial, but identifying positive symbiotic interactions and quantifying the contribution of partners to physiological processes is challenging. For example, our understanding of the relative contribution of sponge cells and their microbial symbionts to the uptake and exchange of dissolved organic matter (DOM)—a major component of sponge diet—is limited. Here, we combined host-symbiont cell separation with pulse-chase isotopic labelling in order to trace the uptake of ¹³ C- and ¹⁵ Nenriched DOM into sponge cells and microbial symbionts of the encrusting Caribbean sponges Haliclona vansoesti and Scopalina ruetzleri , which are low microbial abundance (LMA) species. Sponge cells were responsible for >99% of DOM assimilation during the pulse-chase experiment for both sponge species, while the contribution of symbiotic microbes to total DOM uptake was negligible (<1%). Nitrogen derived from DOM was translocated from sponge cells to microbial cells over time, indicating processing of host nitrogenous wastes by microbial endosymbionts. Thus, host cells drive DOM uptake in these species, while microbial symbionts may aid in the recycling of host-waste products. Our findings highlight the ability of sponges to derive nutrition by internalizing dissolved compounds from their environment and retaining nutrients via host-microbe interactions.
... However, this sponge does appear to consume more detrital matter and POM (Fig. 3) compared to a diet reconstruction from H. caerulea which is likely a reflection of this sponge's unique microbial community and the nutrient cycling processes found within that community. For instance, we do not know whether the microbiome of C. nicholsoni is involved in the uptake of DOM and its re-synthesis and translocation to the host as amino acids as observed in other sponge species (Fiore et al. 2013(Fiore et al. , 2015Shih et al. 2020). It should be noted that the diet reconstruction (Fig. 3) is based on endmember values from other taxa or geographic areas, but no direct feeding measurements were made in this study and none are available from the literature for sclerosponges. ...
... Specifically, in the order Agelasida, where C. nicholsoni is classified, Chloroflexi are often found in high abundances in sponges, generally between 10-29% (Bayer et al. 2018). The metagenomes of Chloroflexi from sponges show wide metabolic diversity that includes the ability to fix carbon under aerobic an anaerobic conditions, conduct ammonia and nitrite uptake, as well as other precursors for multiple biochemical pathways including amino acid biosynthesis (Bayer et al. 2018) which could subsequently be translocated to the host (sensu Shih et al. 2020). In particular, complex carbohydrate degradation pathways have been reported in Chloroflexi, and other members of the prokaryotic microbiome of sponges, which may enhance their ability to utilize the DOM pool on coral reefs (Slaby et al. 2017;Bayer et al. 2018). ...
Article
Full-text available
The sclerosponge Ceratoporella nicholsoni is a hyper-calcifying high microbial abundance sponge. This sponge has been observed at high densities throughout the Caribbean in the mesophotic zone (30–150 m), as well as cryptic environments in shallow (< 30 m) depths. Given the densities of this sponge, it could play an important role in the cycling of inorganic and organic sources of carbon and nitrogen at mesophotic depths. Additionally, there is broad interest in this sponge as a tool for paleobiology, paleoclimatology and paleoceanography. As a result, it is increasingly important to understand the ecology of these unique sponges in the underexplored Caribbean mesophotic zone. Here we show that this sponge increases in abundance from shallow depths into the mesophotic zone of Grand Cayman Island. We observed no significant differences in the stable isotope signatures of δ¹⁵N and δ¹³C of sponge tissue between depths. A predictive model of sponge diet with increasing depth shows that these sponges consume dissolved organic matter of algal and coral origin, as well as the consumption of particulate organic matter consistent with the interpretation of the stable isotope data. The taxonomic composition of the sclerosponge microbiome was invariant across the shallow to mesophotic depth range but did contain the Phylum Chloroflexi, known to degrade a variety of dissolved organic carbon sources. These data suggest that the depth distribution of this sponge may not be driven by changes in trophic strategy and is potentially regulated by other biotic or abiotic factors.
... All three families transport various amino acids as well as phospholipids and heme. The exchange of amino acids between symbiont and sponge host has previously been observed (89) and may provide the Tethybacterales with a competitive advantage over other sympatric microorganisms (90) and possibly allow the sponge hosts to regulate the symbioses via regulation of the quantity of amino acids available for symbiont uptake (91). Similarly, the transfer of heme in the iron-starved ocean environment between sponge host and symbiont could provide a selective advantage, as heme may act as a supply of iron (92). ...
Article
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The fossil record indicates that the earliest evidence of extant marine sponges (phylum Porifera) existed during the Cambrian explosion and that their symbiosis with microbes may have begun in their extinct ancestors during the Precambrian period. Many symbionts have adapted to their sponge host, where they perform specific, specialized functions. There are also widely distributed bacterial taxa such as Poribacteria, SAUL, and Tethybacterales that are found in a broad range of invertebrate hosts. Here, we added 11 new genomes to the Tethybacterales order, identified a novel family, and show that functional potential differs between the three Tethybacterales families. We compare the Tethybacterales with the well-characterized Entoporibacteria and show that these symbionts appear to preferentially associate with low-microbial abundance (LMA) and high-microbial abundance (HMA) sponges, respectively. Within these sponges, we show that these symbionts likely perform distinct functions and may have undergone multiple association events, rather than a single association event followed by coevolution. IMPORTANCE Marine sponges often form symbiotic relationships with bacteria that fulfil a specific need within the sponge holobiont, and these symbionts are often conserved within a narrow range of related taxa. To date, there exist only three known bacterial taxa (Entoporibacteria, SAUL, and Tethybacterales) that are globally distributed and found in a broad range of sponge hosts, and little is known about the latter two. We show that the functional potential of broad-host range symbionts is conserved at a family level and that these symbionts have been acquired several times over evolutionary history. Finally, it appears that the Entoporibacteria are associated primarily with high-microbial abundance sponges, while the Tethybacterales associate with low-microbial abundance sponges.
... These differences might be explained by aerobic microbial processes in HMA sponges, such as nitrification (Hoffmann et al. 2009) or ammonia oxidation (Mohamed et al. 2010), which require O 2 in addition to the O 2 demand based on carbon respiration. Moreover, the organic carbon uptake needed to balance respiration requirements of HMA sponges is potentially further reduced by sponge-associated chemoautotrophs using inorganic carbon sources, which are transferred to the sponge host Pita et al. 2018;Shih et al. 2020). However, van Duyl et al. (2020) found inorganic carbon uptake to represent only 2-3% of deep-sea sponge carbon budgets. ...
Article
Full-text available
Sponges are ubiquitous components of various deep-sea habitats, including cold water coral reefs, and form deep-sea sponge grounds. Although the deep sea is generally considered to be a food-limited environment, these ecosystems are known to be hotspots of biodiversity and carbon cycling. To assess the role of sponges in the carbon cycling of deep-sea ecosystems, we studied the carbon budgets of six dominant deep-sea sponges of different phylogenetic origin, with various growth forms and hosting distinct associated microbial communities, in an ex situ aquarium setup. Additionally, we determined biomass metrics-planar surface area, volume, wet weight, dry weight (DW), ash-free dry weight, and organic carbon (C) content-and conversion factors for all species. Oxygen (O 2) removal rates averaged 3.3 ± 2.8 μmol O 2 g DW sponge h −1 (mean ± SD), live particulate (bacterio-and phytoplankton) organic carbon removal rates averaged 0.30 ± 0.39 μmol C g DW sponge h −1 and dissolved organic carbon (DOC) removal rates averaged 18.70 ± 25.02 μmol C g DW sponge h −1. Carbon mass balances were calculated for four species and revealed that the sponges acquired 1.3-6.6 times the amount of carbon needed to sustain their minimal respiratory demands. These results indicate that irrespective of taxonomic class, growth form, and abundance of microbial symbionts, DOC is responsible for over 90% of the total net organic carbon removal of deep-sea sponges and allows them to sustain themselves in otherwise food-limited environments on the ocean floor.
... Mycale grandis overgrows reef building coral species such as Montipora capitata (Dana 1846) and at least one endemic coral species, Porites compressa (Dana 1846) ). In the specimens we studied, there appears to be a rich mutualistic symbiotic relationship between M. grandis and the microbes they host (Shih et al. 2019). ...
Article
Sponges are ecologically important components of many marine ecosystems and are abundant benthic fauna on coral reefs. Mycale grandis is an alien invasive sponge found on many partially degraded shallow water coral ecosystems in Hawai‘i. Mycale grandis is known to compete spatially with dominant native reef building coral such as Montipora capitata and Porites compressa. Since its appearance in the late 1990s, M. grandis has established itself in a number of coral reef ecosystems around the main Hawaiian Islands. Within south Kāne‘ohe Bay, sponge coverage in 2014–2017 ranged from 2.1% on fringing reefs to 32.3% within the mangrove habitat along the northern edge of Coconut Island, which is similar to coverage found in 2006–2007 surveys. Sponges are prolific filter feeders and pump seawater for the dual purpose of obtaining resources and removing metabolic wastes, and thus process large amounts of water in their environment. Mycale grandis pumps 0.0027 L seawater s–1 L–1 sponge, equivalent to 115 times its own volume per day. These pumping rates were combined with biomass estimates, depth, and circulation parameters in south Kāne‘ohe Bay to show that M. grandis can cycle a substantial amount of the overlying water column and therefore has the potential to influence the biogeochemistry of overlying reef water in south Kāne‘ohe Bay.
... Although we did not detect translocation of microbial-assimilated C and N to the sponge host, the 9-h timeframe may have been insufficient to detect potential nutrient transfer [78]. Translocation of low-molecular weight compounds [79,83] and phagocytosis of microbes appear as the main potential mechanisms for translocation [21]. High rates of phagocytosis in the HMA sponge Geodia barretti are estimated to be sufficient to allow for a significant proportion of microbial-assimilated DOM to be transferred to the host [21], which is consistent with our observations of host cells engulfing symbionts in the HMA sponge. ...
Article
Full-text available
Sponges are the oldest known extant animal-microbe symbiosis. These ubiquitous benthic animals play an important role in marine ecosystems in the cycling of dissolved organic matter (DOM), the largest source of organic matter on Earth. The conventional view on DOM cycling through microbial processing has been challenged by the interaction between this efficient filter-feeding host and its diverse and abundant microbiome. Here we quantify, for the first time, the role of host cells and microbial symbionts in sponge heterotrophy. We combined stable isotope probing and nanoscale secondary ion mass spectrometry to compare the processing of different sources of DOM (glucose, amino acids, algal-produced) and particulate organic matter (POM) by a high-microbial abundance (HMA) and low-microbial abundance (LMA) sponge with single-cell resolution. Contrary to common notion, we found that both microbial symbionts and host choanocyte (i.e. filter) cells and were active in DOM uptake. Although all DOM sources were assimilated by both sponges, higher microbial biomass in the HMA sponge corresponded to an increased capacity to process a gr