Introduction
Benthic foraminifera are ubiquitous marine protists and highly abundant in
coastal sediments
(Lei
et al., 2014; Mojtahid et al., 2016; Murray and Alve, 2000). Coastal
sediments represent the largest pool of marine particulate organic matter
(OM), despite their rather small area (less than 10 % of the ocean floor),
and play an essential role in global carbon and nitrogen cycles
(Jahnke, 2004). Oceanic and terrestrial systems are connected
by the carbon cycling in coastal waters, which contribute to a major part of
the global carbon cycles and budgets
(Bauer
et al., 2013; Cai, 2011; Cole et al., 2007; Regnier et al., 2013). Estuaries
are an important source of organic matter in coastal systems and were
estimated to account for ∼40 % of oceanic phytoplankton
primary productivity (Smith and Hollibaugh, 1993).
Most estuarine areas are considered to be net heterotrophic or act as
carbon sinks (Caffrey,
2003, 2004; Cai, 2011; Herrmann et al., 2015). In general, 30 % of overall
coastal carbon is lost by metabolic oxidation
(Smith and Hollibaugh, 1993). Foraminifera are
highly abundant in estuarine sediments and contribute strongly to these
processes
(Alve
and Murray, 1994; Cesbron et al., 2016; Moodley et al., 2000; Murray and
Alve, 2000). They feed on various sources of labile particulate OM,
including microalgae and detritus, and provide a pivotal link in marine
carbon cycles and food webs
(Bradshaw,
1961; Goldstein and Corliss, 1994; Heinz, 2001; Lee et al., 1966; Lee and
Muller, 1973; Nomaki et al., 2005a, b, 2006, 2009, 2011). The nitrogen
compounds of OM particles are usually remineralized to ammonium
(NH4+). In this way, nitrogen becomes available again as a nutrient for
primary productivity. A major part of this process is attributed to
prokaryotic degraders, but protists are also involved in the process of
regeneration of organic nitrogen compounds
(Ferrier-Pages
and Rassoulzadegan, 1994; Ota and Taniguchi, 2003; Verity et al., 1992). Due
to their high abundances, we consider that foraminifera contribute a large
part to this OM reworking and the regeneration of carbon and nitrogen
compounds from particulate OM sources, e.g., phytodetritus. In this study, we
quantify the bulk OM-derived carbon and nitrogen release, which originates
rather via excretion of organic carbon and nitrogen compounds (vesicular
transport of metabolic waste products), respiration, or diffusion of
inorganic carbon and nitrogen by these single celled microorganisms.
Environmental conditions of temperate tidal flats are physiologically
challenging (high fluctuations of physical and chemical parameters, e.g., temperature and/or OM quality) and therefore host very few, highly adapted
foraminifera species. Monospecific or near monospecific foraminiferal
communities are characteristic of temperate, estuarine regions
(Alve
and Murray, 1994, 2001; Hayward, 2014; Martins et al., 2015; Saad and Wade,
2017). Ammonia tepida and Haynesina germanica are typical representatives of these communities and their
standing crop can reach more than 150 individuals per cm3
(Alve
and Murray, 2001; Mojtahid et al., 2016; Wukovits et al., 2018). Typically,
tidal flats offer a high availability of food sources for phytodetrivores or
herbivores feeding on microalgae. But dense populations of A. tepida communities can
deplete sediments from OM sources and consequently control benthic
meiofaunal community structures
(Chandler,
1989). Therefore, resource partitioning or different metabolic strategies
can be beneficial for foraminifera which share the same spatial and temporal
habitats.
Early experimental investigations and monitoring studies suggest feeding
preferences or selective feeding in littoral foraminifera. However, these
studies rely on indirect observations from environmental monitoring
(Hohenegger et al.,
1989; Papaspyrou et al., 2013) or from a laboratory study focusing on the
more diverse salt marsh communities
(Lee and Muller, 1973). The
latter study revealed that foraminiferal salt marsh communities are
characterized by highly specialized feeding strategies. Analogically, the
close spatial coexistence of A. tepida and H. germanica might also be based on different feeding
strategies and different preferences of other environmental variables. A
major, important difference between the two species subject to this study is
the fact that H. germanica hosts functional plastids derived from ingested microalgae
(Jauffrais
et al., 2016; Lopez, 1979), a phenomenon known as kleptoplasty, which was
first described for a sacoglossan opisthobranch
(Trench, 1969). It was shown that diatom-derived
chloroplasts in the cytoplasm of H. germanica retain their function (as
photosynthetically active kleptoplasts) for up to 2 weeks
(Jauffrais et al.,
2016). Further, there is recent proof that H. germanica takes up inorganic carbon and
nitrogen sources (HCO3 and NH4+) from the surrounding
seawater, most likely to generate metabolites in autotrophic–heterotrophic
interactions with its kleptoplasts (LeKieffre
et al., 2018). Consequently, the mixotrophic lifestyle of H. germanica might lead to a
lower demand of carbon and nitrogen sources and thus to a lower ingestion of
various particulate OM sources as food sources. In contrary, food-derived
chloroplasts in A. tepida lose their photosynthetic activity after a maximum of 24 h (Jauffrais et
al., 2016). Species of the genus Ammonia are described to take up significant
amounts of microalgae and phytodetritus of different origin. Laboratory
feeding experiments have shown that A. tepida responds to several food sources,
including different live microalgae (chlorophytes and diatoms) and
chlorophyte and diatom detritus
(Bradshaw,
1961; LeKieffre et al., 2017; Linshy et al., 2014; Pascal et al., 2008;
Wukovits et al., 2017, 2018), whereas H. germanica shows a low affinity to chloroplast
detritus food sources (Wukovits et al., 2017), but feeds
actively on diatoms (Ward et al.,
2003) and takes up inorganic, dissolved carbon and nitrogen compounds
(LeKieffre et al., 2018). Both species are
found in muddy coastal sediments containing high loads of nutrients or OM
(Armynot du Châtelet et
al., 2009, 2004). But considering their
different feeding strategies, both species might play distinct roles in the
reworking of OM. Recent literature still lacks direct, quantitative
comparisons of foraminiferal species-specific OM-derived C and N ingestion
and release. Therefore, this study aims to compare and quantify variations
in their respective uptake of OM (phytodetritus).
Temperature has a strong impact on metabolic rates and can therefore play
another major role in niche separation or in species-specific adaptations in
the consumer community. Benthic foraminifera show strong metabolic responses
to temperature fluctuations
(Bradshaw,
1961; Cesbron et al., 2016; Heinz et al., 2012). Therefore, seasonal
temperature fluctuations and human-induced global warming can have a strong
impact on foraminiferal community compositions and foraminiferal carbon and
nitrogen fluxes. In estuaries, e.g., temperature acts in many cases as the
most controlling factor on metabolic rates and on net ecosystem metabolism
(Caffrey, 2003). To examine the effect of temperature
on foraminiferal OM processing, temperature variations were included in our
studies. In summary, the aim of this study was to obtain a closer definition
of the ecological feeding niches of A. tepida and H. germanica in relation to intertidal fluxes
of OM and OM processing at different temperatures. Additionally, this study
offers the first estimates for the release of OM-derived carbon and nitrogen
in foraminifera. To reach our aim, we carried out laboratory feeding
experiments with stable-isotope-labeled (13C and 15N) food
sources (chlorophyte detritus: Dunaliella tertiolecta, diatom detritus: Phaeodactylum tricornutum). We compared diatom
detritus intake and retention of phytodetrital carbon (pC) and nitrogen (pN)
of A. tepida and H. germanica at three different temperatures (15, 20,
25 ∘C). The evaluation of the metabolic costs of pC and pN during
a 24 h starvation period can further help to explain species-specific OM
processing due to metabolic nutrient budgets. Further, both food sources
were offered simultaneously to A. tepida to identify feeding preferences of this
species. Finally, we collected quantitative data of the abundances of both
species in the sampling area to estimate species-specific contributions to
intertidal fluxes of OM-derived carbon and nitrogen.
Sampling area.
Material and methods
Sampling area and sample preparation
The sampling area is located at the Elbe river estuary in the German Wadden
Sea (Fig. 1). Samples were collected at low tide in April 2016, close to the
shoreline. Three sediment cores (4.5 cm diameter) were taken in random
spacing within an area of ∼4 m2. The uppermost
centimeter of the cores was fixed with a mixture of ethanol and Rose Bengal
to stain the cytoplasm of live foraminifera. At the University of Vienna,
the sediment core material was sieved to obtain size fractions of 125–250, 250–355, and <355 µm. Brightly
stained (living) foraminifera were identified and counted to calculate
abundances (individuals per m2) to estimate the relevance of A. tepida and H. germanica in
intertidal OM fluxes.
For the laboratory experiments, sediment was collected at low tide from the
uppermost sediment layer and sieved in the field over 125 and 500 µm
to remove larger meiofauna and organic components. Sampling trips
to collect material for laboratory experiments were done in April 2015 and
2016. The sediment was filled into plastic containers with seawater and
transported back to the University of Vienna. The sediment samples were kept
within aquaria, containing filtered water collected at the sampling site.
Foraminifera were picked from the sediment in sufficient number and
collected in crystallizing dishes, containing a layer of North Sea sediment
(<63 µm) and filtered North Sea water (NSW). They were fed
with a mixture of live D. tertiolecta and P. tricornutum once to twice a week until the beginning of the
experiments. Live individuals were identified by showing bright and
intensive cytoplasm color, cyst formation (in case of A. tepida), material gathered
around the aperture, and movement tracks in the sediment. The experiments
started after accumulation of sufficient foraminiferal material 3 weeks
after the field sampling.
Production of artificial phytodetritus
Labeled food was produced by growing D. tertiolecta and P. tricornutum (SAG 1090-1a) in a stable
isotope-enriched growth medium. Algae were cultured in sterile 5 L
Erlenmeyer bottles, containing an F1/2 growth medium (Guillard, 1975; Guillard
and Ryther, 1962) enriched with aliquots of
98 at.%NaH13CO3
and 98 at.%Na15NO3 (SigmaAldrich). The algae culture medium
for Experiment 1 (P. tricornutum) was produced with filtered NSW and enriched with
0.6 mM
NaH13CO3 and 0.9 mM NaNO3 (Na14NO3:Na15NO3→5.25:1), along with the stock solutions for the
F/2 standard protocol. The culture medium for D. tertiolecta (13C single-labeled) in
Experiment 2 was produced with filtered NSW, the stock solutions according
to the F/2 standard protocol, and additionally enriched with 1.5 mM
NaH13CO3 and for P. tricornutum (15N single-labeled) with
1.5 mM
NaHCO3 (natural abundance) and with 0.9 mM NaNO3
(Na14NO3:Na15NO3→5.25:1), along with the stock
solutions for the F/2 standard protocol. The algae cultures were incubated
at 20 ∘C (type ST 2 POL-ECO Aparatura incubation chambers) at a 18h:6h light:dark
cycle and bubbled with ambient air. Cultures were
harvested at stationary growth (after 14–16 days) by centrifugation, washed
three times in sterile, carbon, and nitrogen-free artificial seawater, shock
frozen with liquid nitrogen, and lyophilized to get 13C- and
15N-labeled phytodetritus (cf. Wukovits et
al., 2017). Three batches of algae were produced. Final isotopic
concentrations were P. tricornutum 7 at.%13C and 15 at.%15N (Experiment 1),
D. tertiolecta 22 at.%13C (Experiment 2), and P. tricornutum 14 at.%15N
(Experiment 2).
Experimental setup and conditions.
Species
Individuals
Sampling intervals
T
Food
Amount of food added
Amount of food added
per replicate
(h)
(∘C)
source
(mgCm-2)
(mgNm-2)
Exp. 1
A. tepida
50–55
24/fed
15, 20, 25
Diatom
540
100
24/starved
H. germanica
50–55
24/fed
15, 20, 25
Diatom
540
100
24/starved
Exp. 2
A. tepida
55
1, 3, 6, 12, 24
20
Chlorophyte
410
71
A. tepida
55
1, 3, 6, 12, 24
20
Diatom
647
21
A. tepida
55
1, 3, 6, 12, 24
20
Chlorophyte + diatom
206+324
35+10
Experiment 1: Nutrient demand and temperature response of A. tepida and
H. germanica
A total of 50 to 55 specimens of A. tepida and or H. germanica, respectively, of the size fraction
250–355 µm were distributed into separate wells on a 6-well plate,
containing NSW (12 mL per well, salinity: 28 PSU, practical salinity units,
which lies in the range of our measurements from seawater at the sampling
site: 24–30 PSU). In total, triplicate samples were prepared. The food
source, P. tricornutum (1.5 g dry weight m-2), was added into each well. Wells were
then covered with a headspace to prevent evaporation and were incubated at
15, 20, or 25 ∘C (Table 1).The specimens
were incubated at a 12h:12h light:dark cycle, starting the
incubation with the light cycle. Two equal setups were prepared for
incubation. The first setup was terminated after a 24 h incubation period
to determine the intake of P. tricornutum detritus per species and temperature
(“24 h
fed”). The experimental period of 24 h was chosen to avoid potential
bacterial activity and to maintain system stability. The specimens were
removed from the wells, transferred to Eppendorf© tubes, and frozen
at -20 ∘C. The specimens of the second setup were washed three
times in carbon- and nitrogen-free artificial seawater after the 24 h
incubation period and transferred to crystallizing dishes (9 cm diameter),
containing 150 mL filtered NSW and covered with parafilm. Subsequently, the
dishes were incubated for another 24 h (15, 20, 25 ∘C; 12 h light, 12 h dark, starting with the light
cycle) without food. These samples were analyzed to determine the remaining
phytodetrital carbon and nitrogen after a 24 h starvation period
(“24 h
starved”).
Experiment 2: Feeding preferences of A. tepida
This experiment was carried out at 20 ∘C, since A. tepida specimens
collected in this area showed a good feeding response at this temperature
(Wukovits et al., 2017). Ammonia tepida individuals were incubated at
20 ∘C within 6 well plates (55 individuals per triplicate/well,
size fraction 250–355 µm). Each well was filled with 12 mL NSW.
After acclimation of the individuals within the plates, three different
dietary setups were established (Table 1). The first diet consisted of
chlorophyte-derived detritus, uniformly 13C-labeled (D. tertiolecta, 1.5 g dry
weight cm-2), the second was diatom detritus (P. tricornutum, 1.5 g dry weight
cm-2), uniformly 15N-labeled, and the third consisted of a
homogenized mixture of both food sources (0.73 gcm-2 each). The
differential labeling approach allows calculation of nutrient uptake for
the distinct phytodetritus source after determination of respective algal
carbon and nitrogen composition. Triplicate samples were taken after 1,
3, 6, 12, and 24 h, and specimens were frozen at
-20 ∘C for subsequent isotope (13C/12C and
15N/14N) and elemental analysis (total organic carbon, TOC, and
total nitrogen, TN). Similarly as in Experiment 1, plates were incubated at
a 12h:12h light:dark cycle, starting the incubation with the light
cycle. The algal C:N ratio was used to calculate the pN aliquot for pC of
the 13C-labeled chlorophyte and pC for the 15N-labeled diatom
food source, for a better visual comparison of the food intake (this serves
as a rough estimate of equivalent pC or pN intake in the two diets). This
experiment was solely carried out with A. tepida, since the sediment did not contain
sufficient individuals of H. germanica to set up a parallel run with this species.
Sediment core data and foraminiferal abundances
Sediment core samples (uppermost cm) were sieved to fractionate size classes
(125–250, 250–355, <355 µm). Rose
Bengal-stained individuals were counted for each size fraction to obtain
abundance data for the live foraminiferal community at the sampling date.
Nutrient budget data from the laboratory experiments (individual TOC, TN,
pC, pN), together with the foraminiferal abundances counted from the
sediment cores, were used to estimate the range of foraminiferal
contributions to sedimentary carbon and nitrogen pools and fluxes. In the case
of H. germanica, these contributions were only estimated for the 250–355 µm
fraction (as used in laboratory experiments). For A. tepida, the 125–250 µm
fraction was included in the estimation, using size fraction and feeding
relationships from Wukovits et al. (2018).
Further, the abundances of A. tepida, as derived by the latter study, were compared
with the recent study.
Sample preparation and isotope analysis
Prior to cytoplasm isotope analysis, foraminifera were carefully cleaned
from adhering particles in carbon and nitrogen-free artificial seawater,
rinsed with ultrapure water in a last cleaning step to remove salts,
transferred to tin capsules, and dried at 50 ∘C for several hours.
Subsequently, the foraminifera were decalcified with 10–15 µL 4 % HCl,
and kept at 50 ∘C for 3 days in a final drying step
(Enge
et al., 2014, 2016; Wukovits et al., 2017, 2018). The optimum range for
isotope and elemental analysis was 0.7–1.0 mg cytoplasmic dry weight. In
the 250 µm size fraction, 30–40 individuals met this criterion.
Tools for preparation (hairbrush, needles, tin capsules, tweezers) were
rinsed with dichloromethane (CH2Cl2) and methanol (CH4O)
(1:1, v:v). Glassware for microscopy was combusted at 500 ∘C for
5 h. The samples were analyzed at the Large-Instrument Facility for Advanced
Isotope Research at the University of Vienna (SILVER). Ratios of
13C/12C and 15N/14N and the content of organic carbon and
nitrogen were analyzed with an Isotope Ratio Mass Spectrometer (IRMS;
DeltaPLUS, Thermo Finnigan) coupled with an interface (ConFlo III, Thermo
Finnigan) to an elemental analyzer (EA 1110, CE Instruments). Isotope ratio
data, the Vienna Pee Dee Belemnite standard for C (RVPDB = 0.0112372) and
the standard for atmospheric nitrogen for N (RatmN=0.0036765), were
used to calculate at.% of the samples, where X is 13C or 15N:
at.%X=100×Rstandard×δXsample1000+11+Rstandard×δXsample1000+1.
Intake of pC and pN into foraminiferal cytoplasm was calculated by
determining the excess (E) of isotope content within the samples using
natural abundance data and data of enriched samples
(Middelburg et al.,
2000):
E=at.%Xsample-at.%Xbackground100,
where X is 13C or 15N. Excess and content of total organic carbon
and nitrogen (TOC and TN per individual) were used to calculate incorporated
isotopes (Iiso) derived from the food source:
Iiso=E×TOC (or TN).
The amount of pC (µgind-1) and pN (µgind-1) within
foraminiferal cytoplasm was calculated as follows (Hunter et al., 2012):
pX=Iisoat.%Xphyto100.
Statistical analysis
Experiment 1: The temperature effect on pC and pN within the foraminiferal
cytoplasm, and pC:pN was tested using permutation tests and pairwise
permutation tests for post hoc testing (R package rcompanion). Homogeneity
of variances was tested using the Fligner–Killeen test. Relationships of pC and
pN after feeding and starvation were explored using linear regression for
both species, to observe if pC and pN processing are coupled processes in
the two species. Finally, the relative amount of food-source-derived carbon
and nitrogen after 24 h starvation was evaluated, to compare the
metabolic carbon and nitrogen loss from the two species during the period
without food.
Experiment 2: To describe and compare uptake dynamics for the different
diets, Michaelis–Menten curves were applied on pC and pN data. The models
were tested by applying the lack-of-fit method (R package drc). To compare
pC and pN values for both diets, pN was calculated from pC for D. tertiolecta, and pC from
pN for P. tricornutum. Estimates for pC and pN acquired in this way might be underestimated or
overestimated, respectively, due to possible differences in the ratios of
carbon:nitrogen excretion or remineralization, respectively.
Comparison of pC and pN from diatom feeding in A. tepida and H. germanica after
a 24 h feeding period (24 h fed) and 24 h without food
(24 h
starved) at 15, 20, and 25 ∘C. Letters
show significant differences of (a) cytoplasmic pC, (b) pN between incubation
temperatures within the 24 h feeding period/24 h fed and the
24 h
incubation without food/24 h starved, (c) pC:pN ratio (n=3 in all
cases), and (d) ratios of foraminiferal cytoplasmic C:N; p<0.05,
pairwise permutation tests; ns denotes values that are not significant.
Results
Experiment 1: Nutrient demand and temperature response of A. tepida and
H. germanica
Phytodetrital pC and pN levels derived from P. tricornutum detritus were 2–5 times
higher in A. tepida compared to H. germanica (Fig. 2a, b). Different incubation temperatures
resulted in significant effects on pC levels after 24 h feeding and 24 h
starvation in both species. Ammonia tepida showed a significantly lowered pC content
when feeding at 25 ∘C (Fig. 2a; A. tepida, 24 h fed, p<0.05).
The 24 h incubation period with no food resulted in significantly lowered
pC levels at 20 and 25 ∘C (Fig. 2a; A. tepida,
24 h
starved, p<0.05). In H. germanica, the 24 h feeding period had a similar
effect like on A. tepida, resulting in significantly lowered pC levels at
25 ∘C (Fig. 2a; H. germanica, 24 h fed, p<0.05). A strong effect
of increased temperature after the starvation period was present at
25 ∘C (Fig. 2a; H. germanica, 24 h starved, p<0.05).
The pN levels in A. tepida were considerably affected by temperature after feeding
and starvation, whereas there was no apparent effect on H. germanica pN levels, neither
after feeding, nor after incubation without food (Fig. 2b). Ammonia tepida reacted with
simultaneously lowered pN and pC levels at 25 ∘C after feeding and
starvation (Fig. 2b; A. tepida, p<0.05).
The ratios of pC:pN were affected by temperature in both species during
feeding and starvation (Fig. 2c; p<0.05). Increased temperatures
promoted a drop of pC:pN ratios in A. tepida during the starvation period
(Fig. 2c;
A. tepida, p<0.05). In contrast, temperature-specific pC:pN ratios in H. germanica
showed no change between the incubations with food (24 h fed) and the
starvation period (24 h starved; Fig. 2c; H. germanica). Ratios of C:N show
significant temperature-related changes in H. germanica (p<0.05), but not in
A. tepida (Fig. 2d). The relatively high pN content in A. tepida also shows a steeper
relationship of cytoplasmic pN and pC, compared to H. germanica (Fig. 3a). Further,
there is a far higher metabolic turnover of pC and pN in A. tepida than in H. germanica, specifically at
20 ∘C (Fig. 3b).
(a) Relationship of pC and pN in A. tepida and
H. germanica (A. tepida: R2=0.96, y=1.5x+4.4, p<0.01; H. germanica: R2=0.64, y=x+0.88, p=0.011),
and (b) phytodetrital carbon and nitrogen turnover as percent
release (of total intake of pC or pN per day, respectively).
Comparison of chlorophyte and diatom phytodetritus feeding in
A. tepida for 24 h, presenting feeding dynamics for (a) chlorophyte detritus and
(b) diatom detritus. Curves show Michaelis–Menten fits through triplicates
for each approach (stars indicate calculated values for pC or pN).
Michaelis–Menten parameters of curves for pC and pN intake in
Fig. 4 (bold font shows data from measured values, and regular font shows data
from calculated values; Vmax is the maximum pC/pN, Km is the half saturation for
pC/pN, Res. SE is the residual standard error, and DF is the degrees of freedom).
Vmax
Km
Res. SE
DF
pC
Chlorophyte mono diet
179.875
0.611
20.745
16
Chlorophyte mixed diet
124.196
1.359
11.918
15
Diatom mono diet
80.191
1.374
9.290
16
Diatom mixed diet
24.000
0.098
2.983
16
pN
Chlorophyte mono diet
30.860
0.611
3.559
16
Chlorophyte mixed diet
21.307
1.359
2.286
12
Diatom mono diet
10.912
1.374
1.264
12
Diatom mixed diet
3.267
0.100
0.410
16
Experiment 2: Feeding preferences of A. tepida
Michaelis–Menten curves fitted with no significant deviation of variance
within the sample replicates. Enrichment of algal nutrients in foraminiferal
cytoplasm was highest when a single diet of D. tertiolecta was available (Fig. 4a).
Here, saturation levels (max. 180 ngCind-1) were already reached
within 3 h of detritus introduction, and half saturation with pC
in A. tepida was reached after 0.6 h (Table 2). In contrast, a single P. tricornutum diet
resulted in a slower food intake (Fig. 4b), with a half saturation of pN
levels after 1.4 h (Table 2). Further, diatom phytodetritus intake
resulted in lower levels of pC (max. ∼80 ngCind-1). In
the mixed feeding approach, half saturation of chlorophyte pC was reached
after 1.4 h and diatom pN half saturation was already reached after 0.1 h.
Further, the maximum pC levels of the chlorophyte diet still reached
∼70 % of those in the single chlorophyte diet, whereas the
pN levels of the diatom diet only reached about 30 % of those in the
single diatom diet (Fig. 4, Table 2). Chlorophyte intake was faster and
higher, both in the single and mixed diet, and diatom pN stagnated already
after less than 1 h in the mixed diet, but after this time period,
chlorophyte detritus intake in the mixed diet had continued with increasing
pC levels, saturating between 6 and 10 h (Fig. 4a, b).
Mean abundances (±SD) of live A. tepida and H. germanica (0–1 cm sediment depth),
TOC, TN, and carbon and nitrogen flux calculated from sediment cores (early
May 2015 n=1, late April 2016 n=3). Data for 15 ∘C of
Experiment 1 were used to estimate carbon and nitrogen fluxes (n.d. denotes values that are not
determined).
Size fraction
Abundance
TOC
TN
pCintake
pCrelease
pNintake
pNrelease
(µm)
(indm-2)
(mgm-2)
(mgm-2)
(mgCm-2day-1)
(mgCm-2day-1)
(mgNm-2day-1)
(mgNm-2day-1)
A. tepida
125–250
1 166 979
226.516
77.322
20.937
8.375
5.333
1.813
2015a
250–355
186 742
163.428
35.817
11.467
4.480
1.919
0.651
>355
3773
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
A. tepida
125–250
97 248 (±10471)
21.317
7.277
1.745
0.698
0.444
0.151
2016
250–355
43 594 (±11041)
38.152
8.361
1.802
0.704
0.302
0.102
>355
4401 (±12786)
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
H. germanica
125–250
109 823 (±54078)
25.717
6.867
n.d.
n.d.
n.d.
n.d.
2016
250–355
29 342 (±12768)
30.978
5.311
0.601
0.188
0.069
0.028
>355
3773 (±2741)
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
a Data from Wukovits et al. (2018)
Relevance of A. tepida and H. germanica in intertidal OM
fluxes
Data for the live foraminiferal community in 2016 from the three stained
sediment cores showed a typical, low-biodiversity mudflat community
consisting of A. tepida, H. germanica, and very low abundances of Elphidium williamsonii (<1258 indm-2,
all size fractions). Abundances of A. tepida and H. germanica were equal and decreased with
increasing size fraction. The calculated total biomass of live foraminifera
in units of TOC is max. ∼120 mgCm-2 (both species, all
size fractions; Table 3). From combining in situ abundances and pC values
from Experiment 1 (15 ∘C), this foraminiferal community has the
potential to take up at least 4 mgCm-2day-1, when
taking only diatom detritus into account. The contribution of H. germanica to this OM
processing is only at about 15 %.
Discussion
Different ecologic lifestyles or adaptations to environmental parameters are
important organismic attributes to avoid inter- and intra-specific
competition. Further, different metabolic adaptations result in
species-specific rates of organic matter turnover. Our results clearly
demonstrate that food resource partitioning and different temperature
adaptations contribute to the fluctuating, temporal distribution and
abundance of A. tepida and H. germanica. Due to these specific adaptations, both species play
different roles in intertidal organic matter fluxes. There are, however,
limitations for the interpretation of results derived from laboratory
incubations. A laboratory setup cannot reproduce natural conditions
completely. Therefore, the foraminiferal responses might deviate slightly
from their natural behavior. However, laboratory experiments enable the
analysis of the direct response of specimens to a single factor, while
maintaining other factors at a stable level. To enable a compatible comparison, we
incubated freshly sampled individuals at stable, near-natural conditions.
Both tested food sources are considered good food sources for intertidal
foraminifera (Lee et al., 1966). Dunaliella tertiolecta is commonly used in
feeding experiments with foraminifera due to its easy culturing.
Phaeodactylum tricornutum, which represents a more stable (due to the silicate frustule) source of
OM, is a common food source of intertidal foraminifera (Murray,
1963). Additional tested food sources would give a more comprehensive
picture, but there were limitations in time and material. In the following
sections, our results are discussed with respect to these restrictions.
Experiment 1: Nutrient demand and temperature response of A. tepida and
H. germanica
Experiment 1 shows clear differences in the amount of phytodetritus intake
and different carbon and nitrogen budgeting between the two species (Figs. 2,
3). Ammonia tepida has a higher affinity to the diatom detritus food source with an intake
of diatoms at the two lower temperatures 3 times higher than H. germanica. This lower food intake by H. germanica could be explained by the mixotrophic
lifestyle of this species. Haynesina germanica is known to host kleptoplasts, exploiting the
photosynthetic activity of ingested chloroplasts as an additional energy
source (Lopez, 1979; Pillet et
al., 2011). This species might therefore utilize nutrients (carbohydrates)
derived from the photosynthetic activity of incorporated chloroplasts
(Cesbron et al., 2017). This lifestyle could cause a lower
demand for and lower turnover of OM as food source
(Cesbron et al., 2017). In our study, the pC intake in H. germanica
was ∼67 % lower than that of A. tepida (Fig. 2). Highly specialized
sea slugs use plastids as energy reservoirs at times of low food
availability
(Cartaxana
et al., 2017; Hinde and Smith, 1972; Marín and Ros, 1993), where carbon
supply from chloroplasts can cover 60 % of total carbon input
(Raven et al., 2001). In
kleptoplast-hosting sea slugs, free NH4+ from the seawater is a
primary source of the generation of amino acids via kleptoplast metabolism
within the slug (Teugels et al., 2008). A similar
mechanism in H. germanica might explain the high relative turnover of pN (Fig. 3b).
Phytodetrital nitrogen might therefore be disposed at a higher rate in a
relatively temperature independent process, probably in the form of
dissolved organic nitrogen, further causing a higher pC:pN ratio in the
cytoplasm of H. germanica (Fig. 2).
In addition to the higher rates of phytodetritus intake, A. tepida shows a
considerably higher metabolic turnover of pC and pN than H. germanica (Fig. 3b).
According to Cesbron
et al. (2016), respiration rates (normalized to pmolmm-3day-1) are
about 2–12 times higher in A. tepida specimens than in H. germanica specimens from the same
location. In this study, a 4–7 times higher release of
phytodetritus-derived pC per individual and day (size fraction 250–355 µm)
was observed in A. tepida. Interestingly, this study shows similar
reactions of both species in carbon loss due to increased temperature. An
earlier study on the temperature effect on D. tertiolecta detritus intake of the two
species showed a higher sensitivity to increased temperatures in H. germanica, and far
lower rates of chlorophyte detritus intake compared to this study (Wukovits
et al., 2017). In contrast, A. tepida seems to be more tolerant to higher
temperatures when feeding on chlorophyte detritus. The results of Experiment
1 suggest a niche separation of the two species with respect to
phytodetritus or OM availability and temperature.
Experiment 2: Feeding preferences of A. tepida
The findings of Experiment 2 suggest that A. tepida might prefer OM food sources,
which are easy to exploit and to break down. The high intake values in the
D. tertiolecta mono diet 1 h after incubation and the saturation of cytoplasmic pC
levels after 3 h indicate a high affinity to chlorophyte detritus
(Fig. 4, Table 2). Earlier studies also observed quick and high ingestion
rates of chlorophyte detritus (Chlorella sp.) by the genus Ammonia (Linshy et al., 2014;
Wukovits et al., 2017, 2018). The fast saturation with diatom detritus after
1 h in the mixed diet and the advanced and high intake of D. tertiolecta could even
indicate an avoidance of P. tricornutum and selective feeding on D. tertiolecta. Probably, the soft
cells of chlorophytes enable a faster and easier metabolic processing of
this food source compared to the harder diatom frustules. The recognition of
such food sources could be achieved by chemosensory behavior of the
foraminifera (cf. Langer and Gehring, 1993)
and the attraction to specific substances attached to, or leaking from the
food particles, similar to some other protists, which react to food-specific
amino acids (Almagor et al.,
1981; Levandowsky et al., 1984). Microalgal communities in tidal sediments
typically consist of microphytobenthic diatoms, which are considered to be
the main food source for intertidal foraminifera. An isotope labeling study
has shown that diatoms (Navicula salinicola) are taken up by A. tepida at high rates, but the complete
release of the content of the diatom frustules can take several days
(LeKieffre et al., 2017). This might not
fit the nutrient demands of A. tepida at times of high metabolic activity. Therefore,
a shift from microphytobenthos to particulate OM from riverine or tidal
transport might be a feeding strategy in A. tepida, specifically at higher
temperatures, when more energy is needed to maintain metabolic activities.
In general, food sources of A. tepida include microalgae, phytodetritus, bacteria, and
sometimes metazoans
(Bradshaw,
1961; Dupuy et al., 2010; Moodley et al., 2000; Pascal et al., 2008).
Bacteria are considered to play a minor role in the diet of A. tepida
(Pascal et al., 2008), and reports on
metazoan feeding in A. tepida are restricted to a single observation
(Dupuy et al., 2010). In contrast to A. tepida, H. germanica does
actively ingest bacteria and they can occasionally be preferred over diatoms
(Brouwer et al., 2016). Diatoms
are reportedly taken up by H. germanica, and conical test structures serve as tools to
crack diatom frustules open
(Austin et al.,
2005; Ward et al., 2003). These chloroplasts derived from diatoms remain as
functional kleptoplasts, as mentioned above, within the cytoplasm of H. germanica.
Relevance of A. tepida and H. germanica for intertidal OM
fluxes
Data of foraminiferal abundances or foraminiferal biomass are important
variables to estimate foraminiferal nutrient fluxes. In this section, we
discuss the relevance of A. tepida or H. germanica in intertidal fluxes of phytodetrital carbon
and nitrogen as estimated from sediment core data in combination with
results from the laboratory feeding experiments of this study. The total
biomass of the two species in the sampling area ranges between
∼116 and >380 mgTOCm-2 (size fraction
125–355 µm) at the sampling dates in late April/early May in two
consecutive years (Table 3). This lies within the range of estimations for
hard-shelled foraminifera in other areas of the Wadden Sea
(van Oevelen et
al., 2006a, b; TOC max. ∼160–750 mgCm-2). Our
phytodetritus uptake estimates propose that the foraminiferal biomass
consists of ∼6 %–8 % diatom-derived pC/TOC, with the
major amount contained within A. tepida (compare Table 3). An in situ feeding experiment
with deep-sea foraminifera resulted in values of ∼1 %–12 %
pC/TOC (Nomaki et al., 2005b). Similar in situ incubations in the
core of the oxygen minimum zone of the Arabian Sea report ∼15 % pC/TOC in epifaunal and shallow infaunal foraminiferal carbon uptake
(Enge et al., 2014). In situ incubations offer results closest
to the natural responses of organisms in their natural habitat and enable
precise estimates of foraminiferal nutrient fluxes. Although specific
microhabitat conditions can have a strong influence on organismic behavior, the artificial conditions in laboratory experiments also have an influence
on physiological analysis; therefore the obtained results should be treated
with caution. However, our estimates lie in the same order of magnitude as
the above-mentioned in situ studies and offer a basis for estimations of
foraminiferal carbon and nitrogen fluxes. General variations in
foraminiferal carbon and nitrogen budgets can be caused by different
adaptations to variable food availability in different habitats. This can be
achieved by different controls of energy metabolism
(e.g., Linke, 1992) or
different trophic strategies
(e.g., Lopez, 1979; Nomaki et al., 2011; Pascal et al., 2008). Our results suggest A. tepida has a higher relevance for intertidal OM processing than H. germanica. This can be
mainly attributed to the sequestered chloroplasts within the cytoplasm of
H. germanica. Kleptoplasty is a widespread phenomenon in foraminifera, specifically in
species inhabiting dysoxic sediments, where kleptoplasts could promote
survival in anoxic porewaters (Bernhard and
Bowser, 1999). They might be involved in biochemical pathways within the
foraminiferal cytoplasm, e.g., the transport of inorganic carbon and nitrogen
(LeKieffre et al., 2018). Further,
transmission electron microscopic investigations on H. germanica report a very limited
abundance of food vesicles
(Goldstein and Richardson,
2018). Kleptoplast-bearing species might occupy a distinct niche concerning
their energetic demands. Additionally, they might play an importance in the fluxes of inorganic or dissolved carbon and nitrogen
compounds that has not yet been discovered. However, secondary producers with high uptake rates and a quick
response to particulate OM sources like A. tepida play a strong role in the
biogeochemical carbon and nitrogen recycling.
The high rates of OM carbon and nitrogen turnover are mainly caused by A. tepida
populations (Table 3). The process of carbon and nitrogen regeneration by OM
remineralization might play an important role in marine biogeochemical cycling.
Carbon loss, e.g., due to organismic respiration or OM remineralization to
CO2, reduces the availability of organic carbon sources in the
heterotrophic food web. As mentioned above, in the heterotrophic coastal
zone, 30 % of the carbon pool is lost via respiration, whereas dissolved organic carbon sources from organismic excretion can serve as an
important nutrient source for bacteria
(Kahler
et al., 1997; Snyder and Hoch, 1996; Zweifel et al., 1993). Therefore, the
fast processing of OM in A. tepida might be an important sink for inorganic carbon
(CO2 respiration) and at the same time a link for dissolved organic
carbon sources in intertidal carbon and nitrogen fluxes. According to this
study, the maximum pC flux through A. tepida can reach values of ∼36 mgCm-2day-1
when feeding on chlorophytes at 20 ∘C (estimated
from Experiment 2, Fig. 3 relative release, and max. abundances). Therefore,
A. tepida could contribute up to 10 % of the turnover of OM derived from gross
particulate phytoplankton production on the sampling date in April/May 2016,
with a gross particulate primary production between ∼230 and 1500 mgCm-2day-1
(Tillmann
et al., 2000). This is comparable with the study of
Moodley et al. (2000), in which Ammonia sp. incorporated
∼7 % within 53 h in sediment core incubations' feeding
experiments in sediment incubations with added labeled chlorophyte
detritus.
Planktonic protozoa are the primary regenerators of marine nitrogen,
transforming OM-derived nitrogen to their primary N excretion product,
NH4+ (Glibert, 1997). The excretion of
NH4+ by marine protists can contribute a large part to the
nutritional demands of marine primary productivity (Ferrier-Pages and
Rassoulzadegan, 1994; Ota and Taniguchi, 2003; Verity, 1985). Nitrogen
regeneration by protozoa was supposed to play a far higher role than
bacterial nitrogen regeneration in the marine microbial food chain
(Goldman and Caron, 1985). Indeed, excreted nitrogen
can serve as an important nutrient sources for microbes
(Wheeler and Kirchman, 1986). The release of dissolved
organic nitrogen and NH4+ by, e.g., copepods, can be a major driver
for marine microbial production
(Valdés et al., 2018). Here,
foraminiferal nitrogen excretion values are in the range of estimations for
weight-specific NH4+ excretion in marine protozoa according to
Dolan (1997) (for data for
foraminiferal weight, cf. Supplement Fig. S2). Due to their high
abundances, nitrogen release by A. tepida as observed in this study could reach
2.5 mgNm-2day-1 or ∼73 nmolNdm-2h-1,
respectively, at 15 ∘C and high diatom availability (cf. Table 3).
As a rough estimate for A. tepida feeding at high abundances and high
availability of chlorophyte detritus at 20 ∘C, these values could
increase to ∼22 mgNm-2day-1 or ∼0.6 µmolNdm-2h-1 (Fig. 1, Table 3). Therefore, foraminiferal
nitrogen release as NH4+ or amino acids could cover a considerable
amount of the nutritional nitrogen demand in marine bacteria
(cf. Wheeler and Kirchman, 1986), which assimilate
NH4+ (and amino-acid-derived NH4+) to sustain their
glutamate–glutamine cycle. Vice versa, the labile dissolved organic matter
derived from the bacterial decomposition of refractory organic matter provides a
valuable food source for some benthic foraminifera, and is indispensable for
the reproduction of some foraminiferal species
(Jorissen
et al., 1998; Muller and Lee, 1969; Nomaki et al., 2011). In many marine
diatoms, which are the main drivers of marine primary productivity,
NH4+ is the preferred source of nitrogen uptake over
NO3- (Sivasubramanian and
Rao, 1988). Foraminifera could act as important nutrient providers for
closely associated diatoms, which are also considered as one of their main
food sources (Lee et al., 1966). Consequently, the
kleptoplast-hosting metabolism in H. germanica could benefit from regenerated nitrogen
sources by the high OM mineralization rates in A. tepida. In summary, foraminiferal
carbon and nitrogen fluxes constitute an important link in the food web
complex of primary consumers and decomposers.