Biogeosciences, 13, Author(s) 2016. CC Attribution 3.0 License.
Effect of light on photosynthetic efficiency of sequestered
chloroplasts in intertidal benthic foraminifera
(Haynesina germanica
and Ammonia tepida)
Thierry JBruno JEdouard Metzger, Jean-Luc Mouget, Frans Jorissen, and
Emmanuelle Geslin

1UMR CNRS 6112 LPG-BIAF, Bio-Indicateurs Actuels et Fossiles, Université d'Angers, 2 Boulevard Lavoisier,49045 Angers CEDEX 1, France2EA2160, Laboratoire Mer Molécules Santé, 2 rue de la Houssinière, Université de Nantes, 44322 Nantes CEDEX 3, France3BioISI – Biosystems & Integrative Sciences Institute, Campo Grande University of Lisboa, Faculty of Sciences,1749-016 Lisboa, Portugal4EA2160, Laboratoire Mer Molécules Santé, Université du Maine, Ave O. Messiaen, 72085 Le Mans CEDEX 9, France*These authors contributed equally to this work.
Correspondence to: Thierry Jauffrais ( Received: 21 December 2015 – Published in Biogeosciences Discuss.: 18 January 2016Revised: 22 April 2016 – Accepted: 25 April 2016 – Published: 10 May 2016 Abstract. Some benthic foraminifera have the ability to
account light history. Additionally, this study showed that the incorporate functional chloroplasts from diatoms (klepto- kleptoplasts are unlikely to be completely functional, thus plasty). Our objective was to investigate chloroplast func- requiring continuous chloroplast resupply from foraminifera tionality of two benthic foraminifera (Haynesina german- food source. The advantages of keeping functional chloro- ica and Ammonia tepida) exposed to different irradiance plasts are discussed but more information is needed to better levels (0, 25, 70 µmol photon m−2 s−1) using spectral re- understand foraminifera feeding strategies.
flectance, epifluorescence observations, oxygen evolutionand pulse amplitude modulated (PAM) fluorometry (maxi-mum photosystem II quantum efficiency (Fv/Fm) and rapidlight curves (RLC)). Our results clearly showed that H. germanica was capable of using its kleptoplasts for morethan 1 week while A. tepida showed very limited klep- Benthic foraminifera colonize a wide variety of sediments toplastic ability with maximum photosystem II quantum from brackish waters to deep-sea environments and can be efficiency (Fv/Fm = 0.4), much lower than H. german- the dominant meiofauna in these ecosystems (Gooday, 1986; ica and decreasing to zero in only 1 day. Only H. ger- Pascal et al., 2009). They may play a relevant role in the manica showed net oxygen production with a compensa- carbon cycle in sediments from deep sea (Moodley et al., tion point at 24 µmol photon m−2 s−1 and a production up 2002) to brackish environments (Thibault de Chanvalon et to 1000 pmol O2 cell−1 day−1 at 300 µmol photon m−2 s−1.
al., 2015). Their minor role in organic carbon cycling in aer- Haynesina germanica Fv/Fm slowly decreased from 0.65 to obic sediments, compared to bacteria, contrasts with their 0.55 in 7 days when kept in darkness; however, it quickly de- strong contribution to anaerobic organic matter mineralisa- creased to 0.2 under high light. Kleptoplast functional time tion (Geslin et al., 2011) and they can be responsible for up was thus estimated between 11 and 21 days in darkness and to 80 % of benthic denitrification (Pina-Ochoa et al., 2010; between 7 and 8 days at high light. These results emphasize Risgaard-Petersen et al., 2006).
that studies about foraminifera kleptoplasty must take into Some benthic foraminiferal species are known to sequester chloroplasts from their food source and store them in their Published by Copernicus Publications on behalf of the European Geosciences Union.
T. Jauffrais et al.: Effect of light on photosynthetic efficiency of sequestered chloroplasts
cytoplasm (Lopez, 1979; Bernhard and Bowser, 1999) in a tion is poorly known but longer kleptoplast retention times process known as kleptoplasty (Clark et al., 1990). A klepto- were found in dark treatments (Lopez, 1979; Correia and plast is thus a chloroplast, functional or not, that was "stolen" Lee, 2002b), thus suggesting an effect of light exposure, sim- and integrated by an organism. Kleptoplastic foraminifera ilar to what is observed in kleptoplastic sacoglossans (Trench are found in intertidal sediments (e.g. Haynesina, Elphid- et al., 1972; Clark et al., 1990; Evertsen et al., 2007; Vieira ium and Xiphophaga) (Lopez, 1979; Correia and Lee, 2000, et al., 2009), possibly related to the absence of some compo- 2002a, b; Goldstein et al., 2010; Pillet et al., 2011), low nents of the kleptoplast photosynthetic protein complexes in oxygenated aphotic environments (Nonionella, Nonionel- the host (Eberhard et al., 2008).
lina, Stainforthia) (Bernhard and Bowser, 1999; Grzymski Most recent studies on kleptoplastic foraminifera focused et al., 2002) and shallow-water sediments (Bulimina elegan- on feeding, genetics and microscopic observation related to tissima) (Bernhard and Bowser, 1999). The role of chloro- chloroplast acquisition (e.g., Austin et al., 2005; Pillet et al., plasts sequestered by benthic foraminifera is poorly known 2011; Pillet and Pawlowski, 2013). To our knowledge lit- and photosynthetic functions have only been studied in a tle is known about the effects of abiotic factors on photo- few mudflat species (Elphidium williamsoni, Elphidium ex- synthetic efficiency of sequestered chloroplasts in benthic cavatum and Haynesina germanica) (Lopez, 1979; Correia foraminifera, particularly on the effect of light intensity on and Lee, 2000, 2002a, b; F. Cesbron, personal communica- kleptoplast functionality. Non-invasive techniques are ideal tion, 2015). Amongst the deep-sea benthic foraminifer living to follow photosynthesis and some have already been used to in the aphotic zone, only Nonionella stella has been stud- study foraminifera respiration and photosynthesis, e.g. oxy- ied (Grzymski et al., 2002). The authors suggest that the se- gen evolution by microelectrodes (Rink et al., 1998; Geslin et questered chloroplasts in this species may play a role in the al., 2011) or 14C radiotracer (Lopez, 1979). Recently, pulse assimilation of inorganic nitrogen, even when light is absent.
amplitude modulated (PAM) fluorometry has been used ex- It has also been hypothesised that chloroplast retention may tensively in the study of kleptoplastic sacoglossans (Vieira play a major role in foraminiferal survival when facing star- et al., 2009; Costa et al., 2012; Jesus et al., 2010; Serodio vation periods or in anoxic environments (F. Cesbron, per- et al., 2010; Curtis et al., 2013; Ventura et al., 2013). This sonal communication, 2015). Under these conditions, klepto- non-invasive technique has the advantage of estimating rela- plasts could potentially be used as a carbohydrate source, and tive electron transport rates (rETR) using rapid light curves participate in inorganic nitrogen assimilation (Falkowski and (RLC) and photosystem II (PSII) maximum quantum effi- Raven, 2007) or, when exposed to light, to produce oxygen ciencies (Fv/Fm) very quickly and without incubation peri- needed in foraminiferal aerobic respiration (Lopez, 1979).
ods. The latter parameter has been shown to be a good param- Foraminifera pigment and plastid ultrastructure studies eter to estimate PSII functionality (e.g. Vieira et al., 2009; have shown that the chloroplasts are sequestered from their Jesus et al., 2010; Serodio et al., 2010; Costa et al., 2012; food source, i.e. mainly from diatoms (Lopez, 1979; Knight Curtis et al., 2013; Ventura et al., 2013).
and Mantoura, 1985; Grzymski et al., 2002; Goldstein, The objective of the current work was to investigate the 2004). This was confirmed by experimental feeding stud- effect of irradiance levels on photosynthetic efficiency and ies (Correia and Lee, 2002a; Austin et al., 2005) and by chloroplast functional times of two benthic foraminifera molecular analysis of kleptoplastic foraminifera from differ- feeding in the same brackish areas, H. germanica, which is ent environments (Pillet et al., 2011; Tsuchiya et al., 2015).
known to sequester chloroplasts and A. tepida, not known Foraminifera from intertidal mudflat environments (e.g. H. to sequester chloroplasts. These two species were exposed germanica, A. tepida) feed mostly on pennate diatoms (Pillet to different irradiance levels during 1 week and chloroplast et al., 2011) which are the dominant microalgae in intertidal efficiency was measured using epifluorescence, oxygen mi- mudflat sediments (MacIntyre et al., 1996; Jesus et al., 2009).
crosensors and PAM fluorometry.
Furthermore, in these transitional coastal environments (e.g.
estuaries, bays, lagoons) A. tepida and H. germanica are usu-ally the dominant meiofauna species in West Atlantic French Materials and methods
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 sed-iment is characterised by a clear maximum density at the sur- Haynesina germanica and A. tepida were sampled in January face (Alve and Murray, 2001; Bouchet et al., 2009; Thibault 2015 in Bourgneuf Bay (47.013◦ N, 2.019◦ W), a coastal bay de Chanvalon et al., 2015) with access to light, followed by with a large mudflat situated south of the Loire estuary on the a sharp decrease in the next two centimetres (Thibault de French west coast. In this area, all specimens of A. tepida be- Chanvalon et al., 2015).
long to genotype T6 of Hayward et al. (2004) (M. Schweizer, Foraminiferal kleptoplast retention times can vary from personal communication, 2015). In the field, a large amount days to months (Lopez, 1979; Lee et al., 1988; Correia and (∼ 20 kg) of the upper sediment layer (roughly first 5 mm) Lee, 2002b; Grzymski et al., 2002). The source of this varia- was sampled and sieved over 300 and 150 µm meshes us- Biogeosciences, 13, 2016
T. Jauffrais et al.: Effect of light on photosynthetic efficiency of sequestered chloroplasts
ing in situ sea water. The 150 µm fraction was collected in values of the corresponding epifluorescence photography.
dark flasks and maintained overnight in the dark at 18 ◦C Higher mean pixel values corresponded to foraminifera emit- in the laboratory. No additional food was added. In the fol- ting more fluorescence and thus, as a proxy, contain more lowing day, sediment with foraminifera was diluted with fil- chlorophyll. In an RGB image each channel contains pix- tered (GF/C, 1.2 µm, Whatman) autoclaved sea-water (tem- els between 0 and 255 values. The majority of the infor- perature: 18 ◦C and salinity: 32) and H. germanica and A. mation regarding chlorophyll fluorescence is encoded in the tepida in healthy conditions (i.e. with cytoplasm inside the red channel, therefore the green and blue channel were dis- test) were collected with a brush using a stereomicroscope carded and only the red channel was kept. The images from (Leica MZ 12.5). The selected specimens were rinsed sev- the different treatments were directly comparable as all im- eral times using Bourgneuf bay filtered-autoclaved seawater ages were taken using the same acquisition settings. Thus, to minimize bacterial and microalgal contamination.
the mean red pixel values were used as a proxy for chloro-phyll fluorescence.
Size and biovolume determination
Foraminifera test mean maximal elongation (µm, the lengthof the axes going from the last chamber to the other side Oxygen was measured using advanced Clark type oxy- of the test and passing by the umbilicus) was measured us- gen microelectrodes of 50 µm in diameter (Revsbech, 1989) ing a micrometre mounted on a Leica stereomicroscope (MZ (OXI50 – Unisense, Denmark). Electrodes were calibrated 12.5). Mean foraminiferal volume was approximated with with a solution of sodium ascorbate at 0.1 M (0 %) and with the equation of a half sphere, which is the best resembling seawater saturated with oxygen by bubbling air (100 %).
geometric shape for H. germanica and A. tepida (Geslin et Foraminiferal photosynthesis and oxygen respiration rates al., 2011). The cytoplasmic volume (or biovolume) was then were measured following Høgslund et al. (2008) and Geslin estimated by assuming that the internal test volume corre- et al. (2011). Measurements were carried out in a micro-tube sponds to 75 % of the total foraminiferal test volume (Han- made from glass Pasteur pipette tips with an inner diameter nah et al., 1994).
of 1 mm. The micro-tube was fixed to a small vial, filled withfiltered autoclaved seawater from Bourgneuf Bay. The vial was placed in an aquarium with water kept at room temper-ature (18 ◦C). A small brush was used to position a pool of Pigment spectral reflectance was measured non-invasively 7 to 10 foraminifera in the glass micro-tube after removing to determine and compare the relative pigment composition air bubbles. Oxygen micro-profiles started at a distance of on 50 fresh specimens of H. germanica, on 50 fresh speci- 200 µm above the foraminifers to avoid oxygen turbulences mens of A. tepida and on a benthic diatom as explained in often observed around the foraminifers. Measurements were Jesus et al. (2008). Concisely, a USB2000 (Ocean Optics, registered when the oxygen micro-profiles were stable; they Dunedin, FL, USA) spectroradiometer with a VIS-NIR op- were then repeated five time in the centre of the micro-tube, tical configuration controlled by OObase32 software (Ocean using 50 µm steps until 1000 µm away from the foraminifers Optics B.V., Duiven, the Netherlands) was used. The spec- (Geslin et al., 2011). The oxygen flux (J ) was calculated us- troradiometer sensor was positioned so that the surface was ing the first law of Fick: always viewed from the nadir position. Foraminiferal re- flectance spectra were calculated by dividing the upwelling spectral radiance from the foraminifera (Lu) by the re-flectance of a clean polystyrene plate (Ld) for both of which where D is the oxygen diffusion coefficient (cm2 s−1) at ex- the machine dark noise (Dn) was subtracted (Eq. 1).
perimental temperature (18 ◦C) and salinity (32) (Li and Gre-gory, 1974), and dC/dx is the oxygen concentration gradient 2 cm−1). The O2 concentration gradients were cal- culated with the oxygen profiles and using the R2 of the re-gression line to determine the best gradient. Total O2 con- sumption and production rates were calculated as the prod-uct of O2 fluxes by the surface area of the micro-tube and Foraminifera kleptoplast fluorescence was measured using subsequently divided by the foraminifera number to finally epifluorescence microscopy (× 200, Olympus Ax70 with obtain the cell specific rate (pmol O2 cell−1 d−1) (Geslin et 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 pho- tography) using LUCIA G™ software. The bright field pho-tography was used to trace the contours of the foraminifer All pulse amplitude modulated fluorescence measurements and an ImageJ macro was used to extract the mean pixel were carried out with a Water PAM fluorometer (Walz, Ger- Biogeosciences, 13, 2016
T. Jauffrais et al.: Effect of light on photosynthetic efficiency of sequestered chloroplasts
many) using a blue measuring light. Chloroplast function- fitted with a Haldane model, as modified by Papacek et ality was estimated by monitoring PSII maximum quantum al. (2010) and Marchetti et al. (2013) but without photoin- efficiency (Fv/Fm) and by using P -I rapid light curve (RLC, hibition (Eq. 3).
e.g., Perkins et al., 2006) parameters (α, initial slope of theRLC 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, (pmol O2 cell−1 d−1), I 20, 36, 48, 64, 90 and 128 µmol photons m−2 s−1), each last- (µmol photons m−2 s−1), ing 30 s. The PAM probe was set up on a stand holder at a stant (µmol photons m−2 s−1) and Rd the dark respiration, 2 mm distance from a group of 10 foraminifera.
expressed as an oxygen consumption (pmol O2 cell−1 d−1).
The initial slope of the P -I (Photosynthesis–Irradiance) (pmol O2 cell−1 day−1 (µmol photons m−2 s−1)−1) and the compensation irradiance Haynesina germanica, a species known to sequester chloro- Ic were calculated according to Eqs. (4) and (5).
plasts, were placed in plastic Petri dishes and starved for7 days under three different light conditions: dark (D and Dark-RLC), low light (LL, 25 µmol photons m−2 s−1 high light (HL, 70 µmol photons m−2 s−1); whereas for com- parison, A. tepida, a foraminifer not known to sequester chloroplasts was starved but only exposed to the dark con-dition. A short-term experiment was thus carried out (7 days) Oxygen measurements were repeated at 300 µmol photons to study the effect of light on healthy specimens rather than m−2 s−1 and in the dark at the end of the experiment the effect of starvation. For each condition, 10 specimens (7 days of incubation) for all different light treatments were used per replicate and three replicates per light treat- (D, LL, HL) using 10 specimens, to assess their produc- ment; furthermore all plastic Petri dishes were filled with tion or consumption of oxygen at these two light levels Bourgneuf bay filtered-autoclaved seawater. This experiment (300 µmol photons m−2 s−1 and in the dark) in all treatments.
was carried out in a thermo-regulated culture room at 18 ◦C, For all conditions (D, LL, HL and Dark-RLC) Fv/Fm was equipped with cool light fluorescent lamp (Lumix day light, measured daily at early afternoon, after a 1-hour dark adap- L30W/865, Osram) and using a 14:10 h (Light : Dark) pho- tation period and measurements were done in triplicate for toperiod. The distances between the light and the experimen- each Petri dish.
tal conditions were assessed using a light-metre and a quan- Rapid light curves were also carried out in all light treat- tum sensor (ULM-500 and MQS-B of Walz) to obtain the ments at the beginning and end of the experiment, after 1- desirable light intensities. Concerning the dark condition, the hour dark adaptation for the two tested species. Additionally, Petri dishes were placed in a box covered with aluminium RLC were carried out daily in an extra triplicate kept in the dark (Dark-RLC) throughout the duration of the experiment.
Haynesina germanica kleptoplast fluorescence was mea- sured using epifluorescence microscopy, as explained above, before and after the different light treatments. At the begin- Data are expressed as mean ± standard deviation (SD) when ning of the experiment it was done on 30 independent speci- n = 3 or standard error (SE) when n = 30. Statistical anal- mens to assess the natural and initial variation of Haynesina yses consisted of a t test to compare the foraminifera test germanica kleptoplast fluorescence. At the end of the exper- mean maximal elongation, a non parametric test (Kruskal iment, the measurements were done on all foraminifera ex- Wallis) to compare the mean chlorophyll fluorescence of posed to the different light conditions (a total of 30 speci- the foraminifera exposed to the different experimental con- mens per condition). This was also measured on A. tepida, ditions and a multifactor (experimental conditions (D, LL, but results are not presented because no chlorophyll fluores- HL), irradiance (0–300 µmol photons m−2 s−1)) analysis of cence was observed at the end of the experiment.
variance (ANOVA) with a Fisher's LSD test to compare the Haynesina germanica and A. tepida oxygen production respiration rates at the end of the experiment. Differences and consumption were measured at the beginning of the ex- were considered significant at p < 0.05. Statistical analyses periment on three independent replicates with seven spec- were carried out using the Statgraphics Centurion XV.I (Stat- imens in each replicate. Six different light steps were Point Technologies, Inc.) software.
used to measure O2 production (0, 25, 50, 100, 200 and300 µmol photons m−2 s−1) for H. germanica and only twolight steps (0 and 300 µmol photons m−2 s−1) for A. tep-ida. Photosynthetic activity (P ) data of H. germanica were Biogeosciences, 13, 2016

T. Jauffrais et al.: Effect of light on photosynthetic efficiency of sequestered chloroplasts
Figure 1. Spectral reflectance signatures of Haynesina germanica,
Ammonia tepida and of a benthic diatom in relative units (x axis
legend: Wavelength (nm)).
Figure 2. Illustration of Haynesina germanica chloroplast content
at the beginning (a) and at the end of the experiment for the three ex-
perimental conditions, Dark (b), Low Light (c) and High Light (d).
Size and biovolume
Higher colour scale values correspond to foraminifera emittingmore fluorescence and likely containing more chlorophyll a; flu- Ammonia tepida specimens were larger than H. german- orescence in pixel values between 0 and 255, (scale bar = 50 µm).
ica 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 bio- lis) showing that the differences in chlorophyll a fluores- volumes equal to 1.20 × 107 ± 3.9 × 106 µm3 (SD) and cence were significant (p < 0.01, Df = 3, Fig. 3). It is also 1.01 × 107 ± 3.65 × 106 µm3 (SD), respectively.
noteworthy to mention that there was a large individual vari-ability within each treatment leading to large standard errors in spite of the number of replicates (n = 30).
Fresh Haynesina germanica and A. tepida showed very dif- ferent spectral reflectance signatures (Fig. 1). Haynesina ger- between the two species. Ammonia tepida did not manica showed a typical diatom spectral signature with high show any net oxygen production although respiration reflectance in the infrared region (> 740 nm) and clear ab- rates measured at 300 µmol photons m−2 s−1 were lower sorption features around 585, 630 and 675 nm; the absorption (2485 ± 245 pmol O2 cell−1 d−1) than the ones measured feature around 675 nm corresponds to the presence of chloro- in the dark (3531 ± 128 pmol O2 cell−1 d−1) (F2,2 = 3.7, phyll a; the 585 nm feature is the result of fucoxanthin and p = 0.02). Haynesina germanica showed lower dark res- the 630 nm absorption feature is the result of chlorophyll c piration rates (1654 ± 785 pmol O2 cell−1 d−1) and oxygen (arrows, Fig. 1). Ammonia tepida showed no obvious pig- production quickly increased with irradiance, showing ment absorption features apart from 430 nm (Fig. 1).
no evidence of photoinhibition within the light range Epifluorescence images showed a clear effect of the dif- used (Fig. 4). Compensation irradiance (Ic) was reached ferent light treatments (Dark, Low Light, Hight Light) on H. very quickly, as low as 24 µmol photons m−2 s−1 (95 % germanica chlorophyll fluorescence (Fig. 2). Visual observa- coefficient bound: 17–30 µmol photons m−2 s−1, values cal- tions showed a clear decrease in chlorophyll fluorescence for culated from the fitted model Eq. 4) and the half-saturation the LL and HL treatments from the beginning of the experi- constant (Ek) was also reached at very low light levels, ment (Fig. 2a) to the end of a 7-day period of light exposure i.e. at 17 µmol photons m−2 s−1. No photoinhibition was (Fig. 2c and d, respectively). Samples kept in the dark did not observed under the experimental light conditions (0 to show an obvious decrease but showed a more patchy distribu- 300 µmol photons m−2 s−1), which resulted in an estimation tion compared to the beginning of the experiment (Fig. 2b).
of ∼ 2800 pmol O2 cell−1 d−1 for maximum photosyn- This was confirmed by a non-parametric test (Kruskal Wal- thetic capacity. The P -I curve initial slope at limiting Biogeosciences, 13, 2016
T. Jauffrais et al.: Effect of light on photosynthetic efficiency of sequestered chloroplasts
Table 1. 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).
Respiration rate (pmol O2 cell−1 d−1) Figure 3. Mean chlorophyll a fluorescence (±SE, n = 30) at the end
ent between conditions (p < 0.001), with significantly lower for the three experimental conditions (Dark, Low Light and High respiration rates of specimens incubated under High Light Light) and the beginning (T0) of the experiment using Haynesina conditions than those under Dark and Low Light conditions germanica. Higher mean values likely corresponded to foraminiferacontaining more chlorophyll.
(p < 0.05, Fisher's LSD test).
PAM fluorescence rapid light curve (RLC) parameters (α, rETRmax, Ek and Eopt) showed significant differences be-tween foraminiferal species and over the duration of the ex-periment (Figs. 5 and 6). Highest rETRmax, α and Eopt werealways observed in H. germanica. After only one starvationday A. tepida RLC parameters dropped to zero or close tozero. In contrast, H. germanica RLC parameters showed aslow decrease throughout the experiment (Figs. 5 and 6) withrETRmax and α decreasing from 6 to 4 and 0.22 to 0.15,respectively (Figs. 6a and b). The parameters Ek and Eoptstayed constant over the 7 days of the experiment, with val-ues oscillating around 30 and 90, respectively (Fig. 6c andd).
PSII maximum quantum yields (Fv/Fm) were clearly af- fected by light and time (Fig. 7). Both species showed highinitial Fv/Fm values, i.e. > 0.6 and 0.4 for H. germanica andA. tepida, respectively (Fig. 7). However, while A. tepida Fv/Fm values quickly decreased to zero after only one starva- (pmol O2 cell−1 d−1) as a function of the photon flux density (PFD, tion day, H. germanica exhibited a large variability between µmol photons m−2 s−1). The half-saturation constant, Ek, was light conditions (D, LL, HL) throughout the duration of the found at 17 (13–21), the dark respiration, Rd, at 1654 (1522–1786) experiment (Fig. 7); decreasing from 0.65 to 0.55 in darkness pmol O2 cell−1 d−1 and the maximum photosynthetic capacity, (D), from 0.65 to 0.35 under low light (LL) conditions and Pm, at 2845 (2672–3019) pmol O2 cell−1 d−1. The Ic, calculated from 0.65 to 0.20 under high light (HL). Using these Fv/Fm compensation irradiance (24 (17–30) µmol photons m−2 s−1). Theadjusted R2 of the model was equal to 0.998, n = 3.
decreases, H. germanica kleptoplast functional times wereestimated between 11 and 21 days in the dark (D), 9–12 daysin low light (LL) and 7–8 days in high light (HL), depending irradiance (α) was estimated at 70 pmol O on whether or not an exponential or linear model was applied.
(µmol photons m−2 s−1)−1 (95 % coefficient bound: 58–88).
Ammonia tepida chloroplast functional times were estimated Oxygen measurements carried out at the end of the exper- between 1 and 2 days (exponential and linear model, respec- iment (T7) showed significant different dark and light res- tively) and light exposure reduced the functional time to less piration rates, with light respiration being lower than dark than 1 day (data not shown).
respiration but not reaching net oxygen production rates (D,LL, HL) (Table 1). Moreover, respiration rates were differ- Biogeosciences, 13, 2016
T. Jauffrais et al.: Effect of light on photosynthetic efficiency of sequestered chloroplasts
ida was found to carry out aerobic respiration, but respirationrates measured at 300 µmol photons m−2 s−1 were lower thanthose measured in the dark. We thus suppose that in A. tepidaoxygen production by ingested diatom or chloroplasts mightbe possible, provided that this species is constantly suppliedwith fresh diatoms. However, another possibility to explainthis reduction in oxygen consumption could be a decrease ofits metabolism or activity under light exposure. The light anddark oxygen production or consumption values measured forboth species are in accordance with previous studies (Geslinet 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 (Morvanet al., 2006; Bouchet et al., 2007) and assuming that photo-synthesis produced one mol O2 per mol of C fixed, H. ger- Figure 5. Rapid light curves (RLC, n = 3) expressed as the relative
manica primary production would be between 1.8 × 10−5 electron transport rate (rETR) as a function of the photosynthetic and 4.0 × 10−4 mol C m−2 d−1. This is a very low value active radiation (PAR in µmol photons m−2 s−1) of Haynesina ger- compared to microphytobenthos primary production in At- manica (black lines) and Ammonia tepida (black dashed lines) dur- lantic mudflat ecosystems, which usually range from 1.5 to ing the 7 days of the experiment.
5.9 mol C m−2 d−1 (e.g. Brotas and Catarino, 1995, reviewedin MacIntyre et al., 1996). The estimated values representthus less than 0.1 % of microphytobenthos fixated carbon and are in the same range of values than what has been describedby Lopez (1979) using 14C radioactive tracers. These results should be interpreted with caution because a wide variety offactors probably affect H. germanica in situ primary produc- Our results clearly show that only H. germanica was capable tion, e.g. diatom availability, kleptoplast densities, nutrient of carrying out net photosynthesis. Haynesina germanica had supply, light exposure, sea water turbidity, local biogeochem- typical diatom reflectance spectra (Fig. 1), showing the three ical processes and migration capability are all factors that major diatom pigment absorption features: chlorophyll a, can potentially affect H. germanica kleptoplast functionality.
chlorophyll c, and fucoxanthin (Meleder et al., 2003, 2013; Nevertheless, although carbon fixation seems not to be rele- Jesus et al., 2008; Kazemipour et al., 2012). Conversely, in vant at a global scale, the oxygen production could be impor- A. tepida these absorption features were not detected, sug- tant at a microscale and relevant in local mineralization pro- gesting that diatom pigments ingested by this species were cesses in/on mudflat sediments (e.g. iron, ammonium, man- quickly digested and degraded to a degree where they were no longer detected by spectral reflectance measurements.
At sampling time (T0) H. germanica rETR and Fv/Fm These non-destructive reflectance measurements are thus in values were similar to microphytobenthic species (i.e.
accordance with other studies on benthic foraminifera pig- Fv/Fm > 0.65) (Perkins et al., 2001), suggesting that the klep- ments by HPLC showing that H. germanica feed on benthic toplast PSII and electron transport chain were not much af- diatoms (Knight and Mantoura, 1985). Similarly, Knight and fected after incorporation in the foraminifers' cytoplasm. In Mantoura (1985) also detected higher concentrations and less contrast, A. tepida Fv/Fm and RLC parameters were already degraded diatom pigments in H. germanica than in A. tepida. much lower on the sampling day and quickly decreased to Furthermore, H. germanica has the ability to pro- almost zero within 24 h, suggesting that plastids were not duce oxygen from low to relatively high irradiance, stable inside the A. tepida cytoplasm. Complete diatoms in- as shown by the low compensation point (Ic) of side A. tepida were already observed in feeding studies (Le 24 µmol photons m−2 s−1 and the high onset of light sat- Kieffre, pers. com), this low Fv/Fm value might thus come uration (> 300 µmol photons m−2 s−1) (Fig. 4). Thus, H. from recently ingested diatoms by A. tepida. Fv/Fm has pre- germanica seems to be well adapted to cope with viously been used to determine kleptoplast functional times the high light variability observed in intertidal sedi- and to follow decrease in kleptoplast efficiency in other klep- ments that can range from very high irradiance levels toplastic organisms, e.g. the sea slug Elysia viridis (Vieira (> 1000 µmol photons m−2 s−1) at the surface of the sediment et al., 2009). Fv/Fm measurements carried out on H. ger- during low tide to very low levels within the sediment matrix manica at different light conditions showed that light had a or during high tide in turbid mudflat waters. Ammonia tep- significant effect on the estimation of kleptoplast functional Biogeosciences, 13, 2016
T. Jauffrais et al.: Effect of light on photosynthetic efficiency of sequestered chloroplasts
Figure 6. 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).
time, with the longest functional time estimated at 21 days for was amplified in low and high irradiance and it should dark conditions. This time frame would qualify H. german- be pointed out that the actual light level of the HL treat- ica as a long-term kleptoplast retention species (Clark et al., ment (i.e. 70 µmol photons m−2 s−1) is very low compared 1990); however, our 7 days estimation for the high light treat- to irradiances in their natural environment, which are easily ment would place H. germanica in the medium-term reten- going above 1000 µmol photons m−2 s−1, showing that the tion group. This clearly shows that light exposure has an im- foraminifera kleptoplasts lack the high photoregulation ca- portant effect on this species kleptoplast functionality. Con- pacity exhibited by the benthic diatoms that they feed upon cerning A. tepida, the short dark diatom or chloroplast func- (Cartaxana et al., 2013). This is consistent with the observa- tional time (< 2 days) places this species directly in the short tion at the end of the experiment that no net oxygen produc- or medium-term retention group.
tion was occurring under the different light conditions. Nev- Additionally, H. germanica kept in darkness showed a ertheless, a small difference was still found between dark and slow decrease of the RLC parameters, α and rETRmax, light respiration (Table 1), suggesting that some oxygen pro- throughout the 7 experimental days; this decrease is likely duction was still occurring but it was not sufficient to com- related to overall degradation of the light-harvesting com- pensate for the respiration oxygen consumption. We also no- plexes and of other components of the photosynthetic ap- ticed that the respiration was higher in the foraminifera main- paratus, which gradually induced a reduction of light har- tained in low light and dark conditions in comparison to the vesting efficiency and of carbon metabolism. This decrease high light foraminifera. In the line of the lower Fv/Fm values Biogeosciences, 13, 2016
T. Jauffrais et al.: Effect of light on photosynthetic efficiency of sequestered chloroplasts
This fundamental difference could explain why kleptoplastfunctional times are much longer in N. stella, reaching up to1 year in specimens kept in darkness (Grzymski et al., 2002).
On the other hand, it has been shown that isolated chloro-plasts are able to function for several months in Sacoglossansea slugs provided with air and light in aquaria (Green etal., 2001; Rumpho et al., 2001), which demonstrates the ex-istence of interactions between the kleptoplast and the hostgenomes, and/or of mechanisms facilitating and supportingsuch long-lasting associations. In H. germanica exposed tohigh light it is also possible that reactive oxygen species(ROS) production rates of the sequestered chloroplasts mightexceed the foraminifera capacity to eliminate those ROS,thus inducing permanent damage to the foraminifera. ThisROS production could also eventually damage the klepto-plasts resulting in higher kleptoplast degradation rates.
Figure 7. Maximum quantum efficiency of the photosystem II
Possible advantages of kleptoplasty for intertidal
(Fv/Fm, n = 3) during the experiment for the different applied con- ditions (Dark, Low Light and High Light) and species (Haynesinagermanica and Ammonia tepida).
Much is still unknown about the relationship between klep-toplastic benthic foraminifera and their sequestered chloro- observed, this suggests that kleptoplasts and possibly other plasts. The relevance of the photosynthetic metabolism com- metabolic pathways might have been damaged by the excess pared to predation or organic matter assimilation is unknown; light. Clearly, in H. germanica light exposure had a strong however, it would be of great interest to understand the klep- effect on PSII maximum quantum efficiency and on the re- toplast role in the foraminiferal total energy budget. Oxy- tention of functional kleptoplasts (Fig. 7), which can explain genic photosynthesis comprises multiple reactions leading the absence of net oxygen production after the 7 days of the to the transformation of inorganic carbon to carbohydrates.
experiments. Comparable results for H. germanica were also However, to produce these carbohydrates all the light-driven obtained by counting the number of chloroplasts over time reactions have to be carried out, as well as the Calvin cy- with cells exposed or not to light (Lopez, 1979). One of the cle reactions. With fresh kleptoplasts this hypothesis seems most probable explanations for the observed Fv/Fm decrease possible (e.g. Lopez, 1979), especially if the plastid proteins is the gradual inactivation of the protein D1 in PSII reaction are still present and functional. However, we showed that the centres. This protein is an essential component in the electron maximum quantum efficiency of the PSII decreased quickly transport chain and its turnover rate is frequently the limiting under light exposure, suggesting that substantial direct carbo- factor in PSII repair rates (reviewed in Campbell and Tyys- hydrate production is unlikely without constant chloroplast tjärvi, 2012). Normally, protein D1 is encoded in the chloro- replacement. Conversely, the production of intermediate pho- plast and is rapidly degraded and resynthesized under light tosynthetate products such as adenosine triphosphate (ATP) exposure with a turnover correlated to irradiance (Tyystjärvi and nicotinamide adenine dinucleotide phosphate (NADPH) and Aro, 1996). However, although D1 is encoded by the could be possible and would be of metabolic value for the chloroplast genome, its synthesis and concomitant PSII re- foraminifera. It is also possible that in situ the foraminifera covery require further proteins that are encoded by the algal have better photoregulation capacities. Not only will they nuclear genome (Yamaguchi et al., 2005). Thus, when D1 have easy access to fresh diatom chloroplasts, as H. german- turnover is impaired it will induce an Fv/Fm decrease corre- ica is mainly living in the first few millimetres of the super- lated to irradiance (Tyystjärvi and Aro, 1996) consistent to ficial sediment (Alve and Murray, 2001; Thibault de Chan- what was observed in the present study. In another deep sea valon et al., 2015), but they will also have the possibility of benthic species (Nonionella stella) the D1 and other plastid migrating within the sediment (Gross, 2000) using this be- proteins (RuBisCO and FCP complex) were still present in havioural feature to enhance their photoregulation capacity, the foraminifer 1 year after sampling (Grzymski et al., 2002).
similar to what is observed in benthic diatoms from micro- This shows that some foraminifera can retain both nuclear phytobenthic biofilms (e.g. Jesus et al., 2006; Mouget et al., (FCP) and chloroplast (D1 and RuBisCO) encoded proteins.
2008; Perkins et al., 2010). However, below the photic limit However, contrary to H. germanica, N. stella lives in deeper (max 2 to 3 mm in estuarine sediments reviewed in MacIn- environments never exposed to light and thus is unlikely to tyre et al., 1996; Cartaxana et al., 2011) it is unlikely that carry out oxygenic photosynthesis (Grzymski et al., 2002).
oxygenic photosynthesis will occur, even if live H. german- Biogeosciences, 13, 2016
T. Jauffrais et al.: Effect of light on photosynthetic efficiency of sequestered chloroplasts
ica are also found below this limit (Thibault de Chanvalon et distribution of living benthic foraminifera: First insight from ax- al., 2015; Cesbron et al., 2016).
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REV ARGENT NEUROC VOL. 28, Nº 3 : 78-98 2014 REVISIÓN DE LA LITERATURA Cirugía de los trastornos del comportamiento: el estado del arte Claudio Yampolsky, Damián Bendersky Servicio de Neurocirugía, Hospital Italiano de Buenos Aires, Ciudad Autónoma de Buenos Aires, Argentina RESUmENIntroducción: la cirugía de los trastornos del comportamiento (CTC) se está convirtiendo en un tratamiento más común desde el desarrollo de la neuromodulación. Podemos dividir su historia en 3 etapas: la primera comienza en los inicios de la psicocirugía y termina con el desarrollo de las técnicas estereotácticas, cuando comienza la segunda etapa. Ésta se caracteriza por la realización de lesiones estereotácticas. Nos encontramos transitando la tercera etapa, que comienza cuando la estimulación cerebral profunda (ECP) empieza a ser usada en CTC.Objetivo: el propósito de este artículo es realizar una revisión no sistemática de la historia, indicaciones actuales, técnicas y blancos quirúrgicos de la CTC. Resultados: a pesar de los errores graves cometidos en el pasado, hoy en día, la CTC está renaciendo. Los trastornos psiquiátricos que más frecuentemente se tratan con cirugía y los blancos estereotácticos preferidos para cada uno de ellos son: cápsula interna/estriado ventral para trastorno obsesivo-compulsivo, cíngulo subgenual para depresión y complejo centromediano/parafascicular del tálamo para síndrome de Tourette. Conclusión: los resultados de la ECP en estos trastornos parecen alentadores. Sin embargo, se necesitan más estudios randomizados para establecer la efectividad de la CTC. Debe tenerse en cuenta que una apropiada selección de pacientes nos ayudará a realizar un procedimiento más seguro así como también a lograr mejores resultados quirúrgicos, conduciendo a la CTC a ser más aceptada por psiquiatras, pacientes y sus familias. Se necesita mayor investigación en varios temas como: fisiopatología de los trastornos del comportamiento, indicaciones de CTC y nuevos blancos quirúrgicos.