Introduction
Benthic foraminifera colonize a wide variety of sediments from brackish
waters to deep-sea environments and can be the dominant meiofauna in these
ecosystems (Gooday, 1986; Pascal et al., 2009). They may play a relevant role
in the carbon cycle in sediments from deep sea
(Moodley et al., 2002) to brackish environments
(Thibault de Chanvalon et al., 2015). Their minor role in organic carbon
cycling in aerobic sediments, compared to bacteria, contrasts with their
strong contribution to anaerobic organic matter mineralisation
(Geslin et al., 2011) and they can be responsible for
up to 80 % of benthic denitrification (Pina-Ochoa et al., 2010;
Risgaard-Petersen et al., 2006).
Some benthic foraminiferal species are known to sequester chloroplasts from
their food source and store them in their cytoplasm (Lopez,
1979; Bernhard and Bowser, 1999) in a process known as kleptoplasty (Clark
et al., 1990). A kleptoplast is thus a chloroplast, functional or
not, that was “stolen” and integrated by an organism. Kleptoplastic
foraminifera are found in intertidal sediments (e.g. Haynesina, Elphidium and Xiphophaga)
(Lopez, 1979; Correia and Lee, 2000, 2002a, b; Goldstein et al., 2010; Pillet et al., 2011),
low oxygenated aphotic environments (Nonionella, Nonionellina, Stainforthia) (Bernhard and Bowser, 1999; Grzymski
et al., 2002) and shallow-water sediments (Bulimina elegantissima) (Bernhard and
Bowser, 1999). The role of chloroplasts sequestered by benthic foraminifera
is poorly known and photosynthetic functions have only been studied in a few
mudflat species (Elphidium williamsoni, Elphidium excavatum and Haynesina germanica)
(Lopez, 1979; Correia and Lee, 2000, 2002a, b; F. Cesbron, personal communication, 2015).
Amongst the deep-sea benthic foraminifer living in the
aphotic zone, only Nonionella stella has been studied (Grzymski et al., 2002). The
authors suggest that the sequestered chloroplasts in this species may play a
role in the assimilation of inorganic nitrogen, even when light is absent.
It has also been hypothesised that chloroplast retention may play a major
role in foraminiferal survival when facing starvation periods or in anoxic
environments (F. Cesbron, personal communication, 2015). Under these conditions, kleptoplasts
could potentially be used as a carbohydrate source, and participate in
inorganic nitrogen assimilation (Falkowski and Raven, 2007) or, when
exposed to light, to produce oxygen needed in foraminiferal aerobic
respiration (Lopez, 1979).
Foraminifera pigment and plastid ultrastructure studies have shown that the
chloroplasts are sequestered from their food source, i.e. mainly from
diatoms (Lopez, 1979; Knight and Mantoura, 1985; Grzymski et al., 2002;
Goldstein, 2004). This was confirmed by experimental feeding studies
(Correia and Lee, 2002a; Austin et al., 2005) and by molecular analysis of
kleptoplastic foraminifera from different environments (Pillet et al.,
2011; Tsuchiya et al., 2015). Foraminifera from intertidal mudflat
environments (e.g. H. germanica, A. tepida) feed mostly on pennate diatoms (Pillet et
al., 2011) which are the dominant microalgae in intertidal mudflat sediments
(MacIntyre et al., 1996; Jesus et al., 2009). Furthermore, in these
transitional coastal environments (e.g. estuaries, bays, lagoons) A. tepida and H. germanica are
usually the dominant meiofauna species in West Atlantic French coast
mudflats (Debenay et al., 2000, 2006; Morvan et al., 2006; Bouchet et al.,
2009; Pascal et al., 2009; Thibault de Chanvalon et al., 2015). Their vertical
distribution in the sediment is characterised by a clear maximum density at
the surface (Alve and Murray, 2001; Bouchet et al., 2009; Thibault de
Chanvalon et al., 2015) with access to light, followed by a sharp decrease in
the next two centimetres (Thibault de Chanvalon et al., 2015).
Foraminiferal kleptoplast retention times can vary from days to months
(Lopez, 1979; Lee et al., 1988; Correia and Lee, 2002b; Grzymski et al., 2002).
The source of this variation is poorly known but longer kleptoplast
retention times were found in dark treatments (Lopez, 1979; Correia
and Lee, 2002b), thus suggesting an effect of light exposure, similar to what
is observed in kleptoplastic sacoglossans (Trench et al., 1972; Clark et
al., 1990; Evertsen et al., 2007; Vieira et al., 2009), possibly related to the
absence of some components of the kleptoplast photosynthetic protein
complexes in the host (Eberhard et al., 2008).
Most recent studies on kleptoplastic foraminifera focused on feeding,
genetics and microscopic observation related to chloroplast acquisition
(e.g., Austin et al., 2005; Pillet et al., 2011; Pillet and Pawlowski, 2013).
To our knowledge little is known about the effects of abiotic factors on
photosynthetic efficiency of sequestered chloroplasts in benthic
foraminifera, particularly on the effect of light intensity on kleptoplast
functionality. Non-invasive techniques are ideal to follow photosynthesis
and some have already been used to study foraminifera respiration and
photosynthesis, e.g. oxygen evolution by microelectrodes (Rink et al., 1998;
Geslin et al., 2011) or 14C radiotracer (Lopez, 1979). Recently,
pulse amplitude modulated (PAM) fluorometry has been used extensively in the
study of kleptoplastic sacoglossans (Vieira et al., 2009; Costa et al., 2012;
Jesus et al., 2010; Serodio et al., 2010; Curtis et al., 2013; Ventura et
al.,
2013). This non-invasive technique has the advantage of estimating relative
electron transport rates (rETR) using rapid light curves (RLC) and
photosystem II (PSII) maximum quantum efficiencies (Fv/Fm) very quickly and
without incubation periods. The latter parameter has been shown to be a good
parameter to estimate PSII functionality (e.g. Vieira et al., 2009; Jesus et
al., 2010; Serodio et al., 2010; Costa et al., 2012; Curtis et al., 2013;
Ventura et al., 2013).
The objective of the current work was to investigate the effect of
irradiance levels on photosynthetic efficiency and chloroplast functional
times of two benthic foraminifera feeding in the same brackish areas, H. germanica,
which is known to sequester chloroplasts and A. tepida, not known to sequester
chloroplasts. These two species were exposed to different irradiance levels
during 1 week and chloroplast efficiency was measured using
epifluorescence, oxygen microsensors and PAM fluorometry.
Materials and methods
Sampling
Haynesina germanica and A. tepida were sampled in January 2015 in Bourgneuf
Bay (47.013∘ N, 2.019∘ W), a coastal bay with a large mudflat situated south of
the Loire estuary on the French west coast. In this area, all specimens of
A. tepida belong to genotype T6 of Hayward et al. (2004) (M. Schweizer, personal communication, 2015). In
the field, a large amount (∼ 20 kg) of the upper sediment
layer (roughly first 5 mm) was sampled and sieved over 300 and 150 µm
meshes using in situ sea water. The 150 µm fraction was collected in dark
flasks and maintained overnight in the dark at 18 ∘C in the
laboratory. No additional food was added. In the following day, sediment
with foraminifera was diluted with filtered (GF/C, 1.2 µm, Whatman)
autoclaved sea-water (temperature: 18 ∘C and salinity: 32) and H. germanica
and A. tepida in healthy conditions (i.e. with cytoplasm inside the test) were
collected with a brush using a stereomicroscope (Leica MZ 12.5). The
selected specimens were rinsed several times using Bourgneuf bay
filtered-autoclaved seawater to minimize bacterial and microalgal
contamination.
Size and biovolume determination
Foraminifera test mean maximal elongation (µm, the length of the axes
going from the last chamber to the other side of the test and passing by the
umbilicus) was measured using a micrometre mounted on a Leica
stereomicroscope (MZ 12.5). Mean foraminiferal volume was approximated with
the equation of a half sphere, which is the best resembling geometric shape
for H. germanica and A. tepida (Geslin et al., 2011). The cytoplasmic volume (or biovolume) was
then estimated by assuming that the internal test volume corresponds to
75 % of the total foraminiferal test volume (Hannah et al., 1994).
Spectral reflectance
Pigment spectral reflectance was measured non-invasively to determine and
compare the relative pigment composition on 50 fresh specimens of H. germanica, on 50
fresh specimens of A. tepida and on a benthic diatom as explained in Jesus et al. (2008).
Concisely, a USB2000 (Ocean Optics, Dunedin, FL, USA)
spectroradiometer with a VIS-NIR optical configuration controlled by
OObase32 software (Ocean Optics B.V., Duiven, the Netherlands) was used. The
spectroradiometer sensor was positioned so that the surface was always
viewed from the nadir position. Foraminiferal reflectance spectra were
calculated by dividing the upwelling spectral radiance from the foraminifera
(Lu) by the reflectance of a clean polystyrene plate (Ld) for both of which
the machine dark noise (Dn) was subtracted (Eq. 1).
ρ=(Lu-Dn)(Ld-Dn)
Image analysis
Foraminifera kleptoplast fluorescence was measured using epifluorescence
microscopy (× 200, Olympus Ax70 with Olympus U-RFL-T, excitation
wave length 485 nm). Two Tif images (1232 × 964 px) of each
foraminifer were taken (one bright field photography and one epifluorescence
photography) using LUCIA G™ software. The bright field photography was
used to trace the contours of the foraminifer and an ImageJ macro was used
to extract the mean pixel values of the corresponding epifluorescence
photography. Higher mean pixel values corresponded to foraminifera emitting
more fluorescence and thus, as a proxy, contain more chlorophyll. In an RGB
image each channel contains pixels between 0 and 255 values. The majority of
the information regarding chlorophyll fluorescence is encoded in the red
channel, therefore the green and blue channel were discarded and only the
red channel was kept. The images from the different treatments were directly
comparable as all images were taken using the same acquisition settings.
Thus, the mean red pixel values were used as a proxy for chlorophyll
fluorescence.
Oxygen measurements
Oxygen was measured using advanced Clark type oxygen microelectrodes of 50 µm
in diameter (Revsbech, 1989) (OXI50 – Unisense, Denmark).
Electrodes were calibrated with a solution of sodium ascorbate at 0.1 M
(0 %) and with seawater saturated with oxygen by bubbling air (100 %).
Foraminiferal photosynthesis and oxygen respiration rates were measured
following Høgslund et al. (2008) and Geslin et
al. (2011). Measurements were carried out in a
micro-tube made from glass Pasteur pipette tips with an inner diameter of 1 mm.
The micro-tube was fixed to a small vial, filled with filtered
autoclaved seawater from Bourgneuf Bay. The vial was placed in an aquarium
with water kept at room temperature (18 ∘C). A small brush was
used to position a pool of 7 to 10 foraminifera in the glass micro-tube
after removing air bubbles. Oxygen micro-profiles started at a distance of
200 µm above the foraminifers to avoid oxygen turbulences often
observed around the foraminifers. Measurements were registered when the
oxygen micro-profiles were stable; they were then repeated five time in the
centre of the micro-tube, using 50 µm steps until 1000 µm away
from the foraminifers (Geslin et al., 2011). The oxygen
flux (J) was calculated using the first law of Fick:
J=-D×dCdx,
where D is the oxygen diffusion coefficient (cm2 s-1) at
experimental temperature (18 ∘C) and salinity
(32) (Li and Gregory, 1974), and dC/dx is the oxygen concentration
gradient (pmol O2 cm-1). The O2 concentration gradients were
calculated with the oxygen profiles and using the R2 of the
regression line to determine the best gradient. Total O2 consumption
and production rates were calculated as the product of O2 fluxes by the
surface area of the micro-tube and subsequently divided by the foraminifera
number to finally obtain the cell specific rate (pmol O2 cell-1 d-1)
(Geslin et al., 2011).
Fluorescence
All pulse amplitude modulated fluorescence measurements were carried out
with a Water PAM fluorometer (Walz, Germany) using a blue measuring light.
Chloroplast functionality was estimated by monitoring PSII maximum quantum
efficiency (Fv/Fm) and by using P-I rapid light curve (RLC, e.g., Perkins et al., 2006)
parameters (α, initial slope of the RLC at limiting
irradiance; rETRmax, maximum relative electron transport rate; Ek, light
saturation coefficient; and Eopt, optimum light) (Platt et al.,
1980). Rapid light curves were constructed using eight
incremental light steps (0, 4, 15, 20, 36, 48, 64, 90 and 128 µmol photons m-2 s-1),
each lasting 30 s. The PAM probe was set
up on a stand holder at a 2 mm distance from a group of 10 foraminifera.
Experimental design
Haynesina germanica, a species known to sequester chloroplasts, were placed in plastic Petri
dishes and starved for 7 days under three different light conditions:
dark (D and Dark-RLC), low light (LL, 25 µmol photons m-2 s-1)
and high light (HL, 70 µmol photons m-2 s-1);
whereas for comparison, A. tepida, a foraminifer not known to sequester chloroplasts was
starved but only exposed to the dark condition. A short-term experiment was
thus carried out (7 days) to study the effect of light on healthy specimens
rather than the effect of starvation. For each condition, 10 specimens were
used per replicate and three replicates per light treatment; furthermore all
plastic Petri dishes were filled with Bourgneuf bay filtered-autoclaved
seawater. This experiment was carried out in a thermo-regulated culture room
at 18 ∘C, equipped with cool light fluorescent lamp (Lumix day
light, L30W/865, Osram) and using a 14:10 h
(Light : Dark) photoperiod. The
distances between the light and the experimental conditions were assessed
using a light-metre and a quantum sensor (ULM-500 and MQS-B of Walz) to
obtain the desirable light intensities. Concerning the dark condition, the
Petri dishes were placed in a box covered with aluminium foil.
Haynesina germanica kleptoplast fluorescence was measured using epifluorescence microscopy, as
explained above, before and after the different light treatments. At the
beginning of the experiment it was done on 30 independent specimens to
assess the natural and initial variation of Haynesina germanica kleptoplast fluorescence. At the
end of the experiment, the measurements were done on all foraminifera exposed
to the different light conditions (a total of 30 specimens per condition).
This was also measured on A. tepida, but results are not presented because no
chlorophyll fluorescence was observed at the end of the experiment.
Haynesina germanica and A. tepida oxygen production and consumption were measured at the beginning of
the experiment on three independent replicates with seven specimens in each
replicate. Six different light steps were used to measure O2 production
(0, 25, 50, 100, 200 and 300 µmol photons m-2 s-1) for H. germanica and
only two light steps (0 and 300 µmol photons m-2 s-1) for A. tepida.
Photosynthetic activity (P) data of H. germanica were fitted with a Haldane model, as
modified by Papacek et al. (2010) and Marchetti et al. (2013) but without
photoinhibition (Eq. 3).
P(I)=Pm×II+Ek-Rd,
where Pm is the maximum photosynthetic capacity (pmol O2 cell-1 d-1),
I the photon flux density (µmol photons m-2 s-1),
Ek the half-saturation constant (µmol photons m-2 s-1) and Rd
the dark respiration, expressed as an oxygen consumption (pmol O2 cell-1 d-1).
The initial slope of the P-I (Photosynthesis–Irradiance) curve at limiting irradiance α
(pmol O2 cell-1 day-1 (µmol photons m-2 s-1)-1) and
the compensation irradiance Ic were calculated according to Eqs. (4) and
(5).
Ic=Ek×RdPm-Rdα=RdIc
Oxygen measurements were repeated at 300 µmol photons m-2 s-1
and in the dark at the end of the experiment (7 days of incubation) for all
different light treatments (D, LL, HL) using 10 specimens, to assess their
production or consumption of oxygen at these two light levels (300 µmol photons m-2 s-1
and in the dark) in all treatments.
For all conditions (D, LL, HL and Dark-RLC) Fv/Fm was measured daily at early
afternoon, after a 1-hour dark adaptation period and measurements were
done in triplicate for each Petri dish.
Rapid light curves were also carried out in all light treatments at the
beginning and end of the experiment, after 1-hour dark adaptation for the
two tested species. Additionally, RLC were carried out daily in an extra
triplicate kept in the dark (Dark-RLC) throughout the duration of the
experiment.
Statistical analysis
Data are expressed as mean ± standard deviation (SD) when n= 3 or
standard error (SE) when n= 30. Statistical analyses consisted of a
t test to compare the foraminifera test mean maximal elongation, a non
parametric test (Kruskal Wallis) to compare the mean chlorophyll
fluorescence of the foraminifera exposed to the different experimental
conditions and a multifactor (experimental conditions (D, LL, HL),
irradiance (0–300 µmol photons m-2 s-1)) analysis of variance
(ANOVA) with a Fisher's LSD test to compare the respiration rates at the end
of the experiment. Differences were considered significant at p < 0.05.
Statistical analyses were carried out using the Statgraphics Centurion
XV.I (StatPoint Technologies, Inc.) software.
Results
Size and biovolume
Ammonia tepida specimens were larger than H. germanica with a mean
maximal elongation of 390 ± 42 µm (SD, n= 34) and 366 ± 45 µm
(SD, n= 122), respectively (p < 0.01, F121,33= 1.15). This resulted in
cytoplasmic biovolumes equal to 1.20 × 107 ± 3.9 × 106 µm3
(SD) and 1.01 × 107 ± 3.65 × 106 µm3 (SD), respectively.
Chloroplast functionality
Fresh Haynesina germanica and A. tepida showed very different spectral reflectance signatures (Fig. 1).
Haynesina germanica showed a typical diatom spectral signature with high reflectance in the
infrared region (> 740 nm) and clear absorption features around
585, 630 and 675 nm; the absorption feature around 675 nm corresponds to the
presence of chlorophyll a; the 585 nm feature is the result of fucoxanthin and
the 630 nm absorption feature is the result of chlorophyll c (arrows, Fig. 1).
Ammonia tepida showed no obvious pigment absorption features apart from 430 nm (Fig. 1).
Spectral reflectance signatures of Haynesina germanica, Ammonia tepida and of a benthic diatom in
relative units (x axis legend: Wavelength (nm)).
Epifluorescence images showed a clear effect of the different light
treatments (Dark, Low Light, Hight Light) on H. germanica chlorophyll fluorescence
(Fig. 2). Visual observations showed a clear decrease in chlorophyll
fluorescence for the LL and HL treatments from the beginning of the
experiment (Fig. 2a) to the end of a 7-day period of light exposure
(Fig. 2c and d, respectively). Samples kept in the dark did not show an
obvious decrease but showed a more patchy distribution compared to the
beginning of the experiment (Fig. 2b). This was confirmed by a
non-parametric test (Kruskal Wallis) showing that the differences in
chlorophyll a fluorescence were significant (p < 0.01, Df = 3,
Fig. 3). It is also noteworthy to mention that there was a large
individual variability within each treatment leading to large standard
errors in spite of the number of replicates (n= 30).
Illustration of Haynesina germanica chloroplast content at the beginning (a) and at
the end of the experiment for the three experimental conditions, Dark (b),
Low Light (c) and High Light (d). Higher colour scale values correspond to
foraminifera emitting more fluorescence and likely containing more
chlorophyll a; fluorescence in pixel values between 0 and 255, (scale bar = 50 µm).
Mean chlorophyll a fluorescence (±SE, n= 30) at the end
for the three experimental conditions (Dark, Low Light and High Light) and
the beginning (T0) of the experiment using Haynesina germanica. Higher mean values likely
corresponded to foraminifera containing more chlorophyll.
Oxygen measurements carried out at the beginning of the experiment (T0)
differed considerably between the two species. Ammonia tepida did not show any net oxygen
production although respiration rates measured at 300 µmol photons m-2 s-1
were lower (2485 ± 245 pmol O2 cell-1 d-1)
than the ones measured in the dark (3531 ± 128 pmol O2 cell-1 d-1)
(F2,2= 3.7, p= 0.02). Haynesina germanica showed lower dark
respiration rates (1654 ± 785 pmol O2 cell-1 d-1) and
oxygen production quickly increased with irradiance, showing no
evidence of photoinhibition within the light range used (Fig. 4).
Compensation irradiance (Ic) was reached very quickly, as low as
24 µmol photons m-2 s-1 (95 % coefficient bound:
17–30 µmol photons m-2 s-1, values calculated from the fitted model Eq. 4) and
the half-saturation constant (Ek) was also reached at very low light levels,
i.e. at 17 µmol photons m-2 s-1. No photoinhibition was
observed under the experimental light conditions (0 to 300 µmol photons m-2 s-1), which resulted in an estimation of ∼ 2800 pmol O2 cell-1 d-1
for maximum photosynthetic capacity. The
P-I curve initial slope at limiting irradiance (α) was estimated at
70 pmol O2 cell-1 d-1 (µmol photons m-2 s-1)-1 (95 %
coefficient bound: 58–88).
Net photosynthesis of Haynesina germanica (pmol O2 cell-1 d-1) as a
function of the photon flux density (PFD, µmol photons m-2 s-1).
The half-saturation constant, Ek, was found at 17 (13–21), the
dark respiration, Rd, at 1654 (1522–1786) pmol O2 cell-1 d-1
and the maximum photosynthetic capacity, Pm, at 2845 (2672–3019) pmol O2 cell-1 d-1.
The Ic, calculated compensation irradiance (24
(17–30) µmol photons m-2 s-1).
The adjusted R2 of the model was equal to 0.998, n= 3.
Oxygen measurements carried out at the end of the experiment (T7) showed
significant different dark and light respiration rates, with light
respiration being lower than dark respiration but not reaching net oxygen
production rates (D, LL, HL) (Table 1). Moreover, respiration rates were
different between conditions (p < 0.001), with significantly lower
respiration rates of specimens incubated under High Light conditions than
those under Dark and Low Light conditions (p < 0.05, Fisher's LSD
test).
Light and dark respiration rates (pmol O2 cell-1 d-1) ± SD
of Haynesina germanica in the three experimental conditions (Dark, Low
Light and High Light) at the end of the experiment (Df, degree of freedom,
PFD photon flux density).
Condition
PFD
Respiration rate (pmol O2 cell-1 d-1)
D
300
2452 ± 537
0
3542 ± 765
LL
300
3468 ± 305
0
4015 ± 110
HL
300
1179 ± 261
0
1905 ± 235
Anova
Df
F test
p
Condition
p (α= 0.05)
2
13.1
< 0.001
PFD
p (α= 0.05)
1
5.4
0.026
Interaction
p (α= 0.05)
2
0.3
0.78
PAM fluorescence rapid light curve (RLC) parameters (α, rETRmax, Ek
and Eopt) showed significant differences between foraminiferal species and
over the duration of the experiment (Figs. 5 and 6). Highest rETRmax,
α and Eopt were always observed in H. germanica. After only one starvation day A. tepida
RLC parameters dropped to zero or close to zero. In contrast, H. germanica RLC
parameters showed a slow decrease throughout the experiment (Figs. 5 and
6) with rETRmax and α decreasing from 6 to 4 and 0.22 to 0.15,
respectively (Figs. 6a and b). The parameters Ek and Eopt stayed constant
over the 7 days of the experiment, with values oscillating around 30 and 90,
respectively (Fig. 6c and d).
Rapid light curves (RLC, n= 3) expressed as the relative
electron transport rate (rETR) as a function of the photosynthetic active
radiation (PAR in µmol photons m-2 s-1) of Haynesina germanica (black lines) and
Ammonia tepida (black dashed lines) during the 7 days of the experiment.
Rapid light curve (RLC, n= 3) parameters for Haynesina germanica (Dark-RLC) and
Ammonia tepida maintained in the dark during the experiment, Alpha is the initial slope of
the RLC at limiting irradiance, rETRmax is the maximum relative electron
transport rate, Ek is the light saturation coefficient and Eopt is the
optimum light, all of them were estimated by adjusting the experimental data
to fit the model of Platt et al. (1980).
PSII maximum quantum yields (Fv/Fm) were clearly affected by light and
time (Fig. 7). Both species showed high initial Fv/Fm values, i.e. > 0.6
and 0.4 for H. germanica and A. tepida, respectively (Fig. 7).
However, while A. tepida Fv/Fm values quickly
decreased to zero after only one starvation day, H. germanica exhibited a large
variability between light conditions (D, LL, HL) throughout the duration of
the experiment (Fig. 7); decreasing from 0.65 to 0.55 in darkness (D),
from 0.65 to 0.35 under low light (LL) conditions and from 0.65 to 0.20
under high light (HL). Using these Fv/Fm decreases, H. germanica kleptoplast functional times
were estimated between 11 and 21 days in the dark (D), 9–12 days in low light
(LL) and 7–8 days in high light (HL), depending on whether or not an exponential or linear
model was applied. Ammonia tepida chloroplast functional times were estimated between 1 and 2 days (exponential and linear model, respectively) and light exposure reduced
the functional time to less than 1 day (data not shown).
Maximum quantum efficiency of the photosystem II (Fv/Fm, n= 3)
during the experiment for the different applied conditions (Dark, Low Light
and High Light) and species (Haynesina germanica and Ammonia tepida).
Discussion
Chloroplast functionality
Our results clearly show that only H. germanica was capable of carrying out net
photosynthesis. Haynesina germanica had typical diatom reflectance spectra (Fig. 1), showing
the three major diatom pigment absorption features: chlorophyll a,
chlorophyll c, and fucoxanthin (Meleder et al., 2003, 2013; Jesus et al., 2008;
Kazemipour et al., 2012). Conversely, in A. tepida these
absorption features were not detected, suggesting that diatom pigments
ingested by this species were quickly digested and degraded to a degree
where they were no longer detected by spectral reflectance measurements.
These non-destructive reflectance measurements are thus in accordance with
other studies on benthic foraminifera pigments by HPLC showing that H. germanica feed on
benthic diatoms (Knight and Mantoura, 1985). Similarly, Knight and Mantoura (1985) also detected higher concentrations and less degraded diatom pigments
in H. germanica than in A. tepida.
Furthermore, H. germanica has the ability to produce oxygen from low to relatively high
irradiance, as shown by the low compensation point (Ic) of 24 µmol photons m-2 s-1
and the high onset of light saturation
(> 300 µmol photons m-2 s-1) (Fig. 4). Thus, H. germanica
seems to be well adapted to cope with the high light variability observed in
intertidal sediments that can range from very high irradiance levels
(> 1000 µmol photons m-2 s-1) at the surface of
the sediment during low tide to very low levels within the sediment matrix
or during high tide in turbid mudflat waters. Ammonia tepida was found to carry out aerobic
respiration, but respiration rates measured at 300 µmol photons m-2 s-1 were lower than those measured in the dark. We thus
suppose that in A. tepida oxygen production by ingested diatom or chloroplasts might
be possible, provided that this species is constantly supplied with fresh
diatoms. However, another possibility to explain this reduction in oxygen
consumption could be a decrease of its metabolism or activity under light
exposure. The light and dark oxygen production or consumption values
measured for both species are in accordance with previous studies (Geslin et
al., 2011).
According to Lopez (1979), measured oxygen data can be used to estimate H. germanica
carbon fixation rates. Thus, using 1000 pmol O2 cell-1 d-1 at
300 µmol photons m-2 s-1, ∼ 200 to 4000 cells
per 50 cm3 in the top 0.5 cm (Morvan et al., 2006; Bouchet et al., 2007)
and assuming that photosynthesis produced one mol O2 per mol of C
fixed, H. germanica primary production would be between 1.8 × 10-5 and
4.0 × 10-4 mol C m-2 d-1. This is a very low value
compared to microphytobenthos primary production in Atlantic mudflat
ecosystems, which usually range from 1.5 to 5.9 mol C m-2 d-1
(e.g. Brotas and Catarino, 1995, reviewed in MacIntyre et al., 1996). The
estimated values represent thus less than 0.1 % of microphytobenthos
fixated carbon and are in the same range of values than what has been
described by Lopez (1979) using 14C radioactive tracers. These results
should be interpreted with caution because a wide variety of factors
probably affect H. germanica in situ primary production, e.g. diatom availability, kleptoplast
densities, nutrient supply, light exposure, sea water turbidity, local
biogeochemical processes and migration capability are all factors that can
potentially affect H. germanica kleptoplast functionality. Nevertheless, although carbon
fixation seems not to be relevant at a global scale, the oxygen production
could be important at a microscale and relevant in local mineralization
processes in/on mudflat sediments (e.g. iron, ammonium, manganese).
At sampling time (T0) H. germanica rETR and Fv/Fm values were similar to microphytobenthic
species (i.e. Fv/Fm > 0.65) (Perkins et al., 2001), suggesting that the
kleptoplast PSII and electron transport chain were not much affected after
incorporation in the foraminifers' cytoplasm. In contrast, A. tepida Fv/Fm and RLC
parameters were already much lower on the sampling day and quickly decreased
to almost zero within 24 h, suggesting that plastids were not stable
inside the A. tepida cytoplasm. Complete diatoms inside A. tepida were already observed in
feeding studies (Le Kieffre, pers. com), this low Fv/Fm value might thus come
from recently ingested diatoms by A. tepida. Fv/Fm has previously been used to determine
kleptoplast functional times and to follow decrease in kleptoplast
efficiency in other kleptoplastic organisms, e.g. the sea slug Elysia viridis (Vieira et
al., 2009). Fv/Fm measurements carried out on H. germanica at different light conditions
showed that light had a significant effect on the estimation of kleptoplast
functional time, with the longest functional time estimated at 21 days for
dark conditions. This time frame would qualify H. germanica as a long-term kleptoplast
retention species (Clark et al., 1990); however, our 7 days estimation
for the high light treatment would place H. germanica in the medium-term retention
group. This clearly shows that light exposure has an important effect on
this species kleptoplast functionality. Concerning A. tepida, the short dark diatom or
chloroplast functional time (< 2 days) places this species directly
in the short or medium-term retention group.
Additionally, H. germanica kept in darkness showed a slow decrease of the RLC
parameters, α and rETRmax, throughout the 7 experimental days;
this decrease is likely related to overall degradation of the
light-harvesting complexes and of other components of the photosynthetic
apparatus, which gradually induced a reduction of light harvesting
efficiency and of carbon metabolism. This decrease was amplified in low
and high irradiance and it should be pointed out that the actual light level
of the HL treatment (i.e. 70 µmol photons m-2 s-1) is very
low compared to irradiances in their natural environment, which are
easily going above 1000 µmol photons m-2 s-1, showing that
the foraminifera kleptoplasts lack the high photoregulation capacity
exhibited by the benthic diatoms that they feed upon (Cartaxana et al.,
2013). This is consistent with the observation at the end of the experiment
that no net oxygen production was occurring under the different light
conditions. Nevertheless, a small difference was still found between dark
and light respiration (Table 1), suggesting that some oxygen production was
still occurring but it was not sufficient to compensate for the respiration
oxygen consumption. We also noticed that the respiration was higher in the
foraminifera maintained in low light and dark conditions in comparison to
the high light foraminifera. In the line of the lower Fv/Fm values observed, this
suggests that kleptoplasts and possibly other metabolic pathways might have
been damaged by the excess light. Clearly, in H. germanica light exposure had a
strong effect on PSII maximum quantum efficiency and on the retention of
functional kleptoplasts (Fig. 7), which can explain the absence of net
oxygen production after the 7 days of the experiments. Comparable results
for H. germanica were also obtained by counting the number of chloroplasts over time
with cells exposed or not to light (Lopez, 1979). One of the most probable
explanations for the observed Fv/Fm decrease is the gradual inactivation of the
protein D1 in PSII reaction centres. This protein is an essential component
in the electron transport chain and its turnover rate is frequently the
limiting factor in PSII repair rates (reviewed in Campbell and
Tyystjärvi, 2012). Normally, protein D1 is encoded in the chloroplast and
is rapidly degraded and resynthesized under light exposure with a turnover
correlated to irradiance (Tyystjärvi and Aro, 1996). However, although D1
is encoded by the chloroplast genome, its synthesis and concomitant PSII
recovery require further proteins that are encoded by the algal nuclear
genome (Yamaguchi et al., 2005). Thus, when D1 turnover is impaired it will
induce an Fv/Fm decrease correlated to irradiance (Tyystjärvi and Aro, 1996)
consistent to what was observed in the present study. In another deep sea
benthic species (Nonionella stella) the D1 and other plastid proteins (RuBisCO and FCP
complex) were still present in the foraminifer 1 year after sampling
(Grzymski et al., 2002). This shows that some foraminifera can retain both
nuclear (FCP) and chloroplast (D1 and RuBisCO) encoded proteins. However,
contrary to H. germanica, N. stella lives in deeper environments never exposed to light and thus
is unlikely to carry out oxygenic photosynthesis (Grzymski et al., 2002).
This fundamental difference could explain why kleptoplast functional times
are much longer in N. stella, reaching up to 1 year in specimens kept in darkness
(Grzymski et al., 2002). On the other hand, it has been shown that isolated
chloroplasts are able to function for several months in Sacoglossan sea
slugs provided with air and light in aquaria (Green et al., 2001; Rumpho et
al., 2001), which demonstrates the existence of interactions between the
kleptoplast and the host genomes, and/or of mechanisms facilitating and
supporting such long-lasting associations. In H. germanica exposed to high light it is
also possible that reactive oxygen species (ROS) production rates of the
sequestered chloroplasts might exceed the foraminifera capacity to eliminate
those ROS, thus inducing permanent damage to the foraminifera. This ROS
production could also eventually damage the kleptoplasts resulting in higher
kleptoplast degradation rates.
Possible advantages of kleptoplasty for intertidal benthic
foraminifera
Much is still unknown about the relationship between kleptoplastic benthic
foraminifera and their sequestered chloroplasts. The relevance of the
photosynthetic metabolism compared to predation or organic matter
assimilation is unknown; however, it would be of great interest to
understand the kleptoplast role in the foraminiferal total energy budget.
Oxygenic photosynthesis comprises multiple reactions leading to the
transformation of inorganic carbon to carbohydrates. However, to produce
these carbohydrates all the light-driven reactions have to be carried out,
as well as the Calvin cycle reactions. With fresh kleptoplasts this
hypothesis seems possible (e.g. Lopez, 1979), especially if the plastid
proteins are still present and functional. However, we showed that the
maximum quantum efficiency of the PSII decreased quickly under light
exposure, suggesting that substantial direct carbohydrate production is
unlikely without constant chloroplast replacement. Conversely, the
production of intermediate photosynthetate products such as adenosine
triphosphate (ATP) and nicotinamide adenine dinucleotide phosphate (NADPH)
could be possible and would be of metabolic value for the foraminifera. It
is also possible that in situ the foraminifera have better photoregulation
capacities. Not only will they have easy access to fresh diatom
chloroplasts, as H. germanica is mainly living in the first few millimetres of the superficial
sediment (Alve and Murray, 2001; Thibault de Chanvalon et al., 2015), but they
will also have the possibility of migrating within the sediment (Gross, 2000)
using this behavioural feature to enhance their photoregulation capacity,
similar to what is observed in benthic diatoms from microphytobenthic
biofilms (e.g. Jesus et al., 2006; Mouget et al., 2008; Perkins et al., 2010).
However, below the photic limit (max 2 to 3 mm in estuarine sediments
reviewed in MacIntyre et al., 1996; Cartaxana et al., 2011) it is unlikely
that oxygenic photosynthesis will occur, even if live H. germanica are also found below
this limit (Thibault de Chanvalon et al., 2015; Cesbron et al., 2016).