The effect of salinity, light regime and food source on C and N uptake in a kleptoplast-bearing 1 foraminifera 2

10 Foraminifera are unicellular organisms that play an important role in marine organic matter cycles. Some species are 11 able to isolate chloroplasts from their algal food source and incorporate them as kleptoplasts into their own metabolic 12 pathways, a phenomenon known as kleptoplastidy. One species showing this ability is Elphidium excavatum , a common 13 foraminifer in the Kiel fjord, Germany. The Kiel fjord is fed by several rivers and thus forms a habitat with strongly 14 fluctuating salinity. Here, we tested the effects of food source, salinity and light regime on the food uptake (via 15 N and 15 13 C algal uptake) in this kleptoplast-bearing foraminifer. In our study E. excavatum was cultured in the lab at three 16 salinity levels (15, 20, 25 PSU) and uptake of C and N from the food source Dunaliella tertiolecta (Chlorophyceae) and 17 Leyanella arenaria (Bacillariophyceae) were measured over time (after 3, 5, 7 days). The species was very well adapted 18 to the current salinity of the sampling region, as both, algal N and C uptake was highest at 20 PSU. It seems that E. 19 excavatum coped better with lower than with higher salinities. The amount of absorbed C from the green algae D. 20 tertiolecta showed a marginal significant effect of salinity, peaking at 20 PSU. Nitrogen uptake was also highest at 20 21 PSU and steadily increased with time. In contrast, C uptake from the diatom L. arenaria was highest at 15 PSU and 22 decreased at higher salinities. We found no overall significant differences in C and N uptake from green algae versus 23 diatoms. Furthermore, the food uptake at a light/dark rhythm of 16:8 h was compared to continuous darkness. Darkness 24 had a negative influence on algal C and N uptake, and this effect increased with incubation time. Starving experiments 25 showed a stimulation of food uptake after 7 days. In summary, it can be concluded that E. excavatum copes well with 26 changes of salinity to a lower level. For changes in light regime, we showed that light reduction caused a decrease of C 27 and N uptake by E. excavatum . species Ammonia tepida increased with salinity (Lintner et al., 2020). In the same study, both species showed large differences in the retention of C relative to N, with subsequent adverse effects on the re-cycling of these elements by mineralization/respiration and excretion to the environment. Such differences, given that these species are (co)dominant 77 in their foraminifera community, can have important implications on local marine biogeochemical cycles of C and N. Based on the above mentioned aspects, this study investigated the food uptake and food preference (green algae versus 79 diatoms) of Elphidium excavatum ssp. at different salinity levels and a changing light/dark rhythm. Elphidium excavatum 80 is optimally suited for this purpose, as it is representative for foraminifera in coastal regions and can account for over 81 90% of the total foraminiferal population in some areas (Schönfeld and Numberger, 2007). Wukovits et al. 2017), indicating that the chloroplasts can supplement the C nutrition of species exhibiting kleptoplastidy. A shift in food preference in terms of C uptake from diatoms at 15 PSU to green algae at 20-25 PSU is also noteworthy and has not yet been observed in this or other foraminifera species. This might have strong implications on foraminiferal C and N re- cycling in habitats where E. excavatum is dominant, given that N retention was approximately 3-fold higher with diets of green algae compared to diatoms (pC:pN 2.2-2.7 for D. tertiolecta compared to 6.4-7.5 for L. arenaria ). On a closer look, it can be seen that foraminifera reacted to an increased salt content in the longer term by lower rates of green algal food consumption. The mean C uptake recorded at 20 PSU showed a maximum five days after food addition and declined thereafter. Such a behavior is already known from H. germanica (Lintner et al., 2020), a closely related species living in the same habitat. In Lintner et al. (2020) this behavior was explained by the fact that H. germanica also 280 contained kleptoplasts, which may serve as internal C and N sources via digestion. In the case of foraminiferal N uptake in our study this effect was not evident, as the amount of incorporated N increased steadily, at least at 15 and 20 PSU. At


Introduction 30
1.1. General information 31 Foraminifera are unicellular, highly diverse marine organisms known since the early Cambrian (e.g., Scott et al., 2003; 32 Pawlowski et al., 2003). As major consumers of phytodetritus they play an important role in organic matter recycling in 33 marine environments, particularly in marine sediments (benthos), from coasts to the deep sea, and in brackish water foraminifer (E. e. excavatum and E. e. clavatum) have been found to coexist in the Baltic Sea (Lutze, 1965

58
Further experiments showed that not all algae are excellent chloroplast donors (Lee and Lee, 1989; Correia and Lee, 59 2001). It was observed that Elphidium absorbs up to five times more chloroplasts from diatoms than from green algae 60 (Correia and Lee, 2000). It was also pointed out that different light/dark regimes had no influence on the uptake of 61 chloroplasts by Elphidium (Correia and Lee, 2000). Foraminifera below the photic zone can also perform kleptoplastidy species Ammonia tepida increased with salinity (Lintner et al., 2020). In the same study, both species showed large 75 differences in the retention of C relative to N, with subsequent adverse effects on the re-cycling of these elements by 76 mineralization/respiration and excretion to the environment. Such differences, given that these species are (co)dominant 77 in their foraminifera community, can have important implications on local marine biogeochemical cycles of C and N.

78
Based on the above mentioned aspects, this study investigated the food uptake and food preference (green algae versus 79 diatoms) of Elphidium excavatum ssp. at different salinity levels and a changing light/dark rhythm. Elphidium excavatum 80 is optimally suited for this purpose, as it is representative for foraminifera in coastal regions and can account for over 81 90% of the total foraminiferal population in some areas (Schönfeld and Numberger, 2007  In the southeast of the Fjord, a fresh water supply, the Schwentine, contributes to a lower salinity of water in this 91 area. Earlier investigations showed that occasional sea water inflow from the Baltic Sea (very saline surface water with 92 33 PSU) has no major impact on the hydrography in the Kiel Fjord (Fennel, 1996). The most common sediments in the 93 fjord are fine sand and dark, organic rich mud (especially found in the inner Fjord). In this area corrosion (abrasion and 94 redeposition) of foraminiferal tests plays an important role, due to the undersaturation of carbonate in the surface water 95 (Grobe and Fütterer, 1981 (Senocak, 1995;ter Jung, 1992). Furthermore, the Kiel Fjord is rich in nutrients and 99 organic C. This accumulation of nutrients originates from the city or the surrounding industrial areas and causes a strong 100 eutrophication in the inner Fjord (Gerlach, 1984). The high input of nutrients leads to a high primary production which,

127
The C:N ratios based on C and N content of the diatom and the green algal food source were 9.14 for L.

166
Since the heavy stable isotopes used as a tracer ( 13 C and 15 N) are also occurring naturally, the natural abundance of these 167 isotopes needs to be accounted for which was measured in foraminifera that did not obtain labelled algal food sources. To 168 take this into account, the so-called isotope excess (E) is calculated (Middelburg et al., 2000): 169 . (2)

170
As Xbackground isotope abundances of foraminifera were used, which were not fed and thus reflect the natural isotope 171 abundance signal.

172
The absorbed amount of isotopes can now be quantified, i.e. labeled Iiso for incorporated C or N.
Here, either the number of individuals (ind -1 ) or the mass (dry matter without test, see 3.

181
To test the main effects of salinity, food source, time, dark: light cycles and starvation, as well as their interaction, on pC 182 and pN uptake we applied two-way and three-way analysis of variance (ANOVA, 95,0 % confidence intervals). Data 183 were log transformed when they did not meet normality or homoscedasticity. If the data were significant a Fisher´s LSD 184 post hoc test was used for more detailed analysis. All statistical tests were performed using Statgraphics Centurion XVI.

185
The points in the graphs are the mean values from triplicates, with an 2σ error bar for the standard deviation.

254
The longer the foraminifera were starved, the more food was consumed within 24 h (Fig. 3)

276
On a closer look, it can be seen that foraminifera reacted to an increased salt content in the longer term by lower rates of 277 green algal food consumption. The mean C uptake recorded at 20 PSU showed a maximum five days after food addition 278 and declined thereafter. Such a behavior is already known from H. germanica (Lintner et al., 2020), a closely related 279 species living in the same habitat. In Lintner et al. (2020) this behavior was explained by the fact that H. germanica also 280 contained kleptoplasts, which may serve as internal C and N sources via digestion. In the case of foraminiferal N uptake 281 in our study this effect was not evident, as the amount of incorporated N increased steadily, at least at 15 and 20 PSU. At this point it should be noted that foraminifera metabolize food C and N during their digestive process and release them 283 into the surrounding environment as excreta or as respiratory CO2 (Hannah et al., 1994;Nomaki et al., 2014). This needs 284 to be taken into account the longer an experiment lasts and might explain the decrease in the incorporated amount of C 285 from day 5 to 7 (Fig. 1). Although C is constantly being absorbed by foraminifera in the form of food, it is also partially 286 relocated and excreted or released by cellular respiration (Hannah et al., 1994). Furthermore, C can also be used for test 287 formation. During the preparation of foraminifera for isotope analysis, the test is dissolved in hydrochloric acid and the 288 amount of incorporated C in the test is not measured, which may cause an underestimation of pC relative to pN at   . 2). Foraminifera had a much lower C and N uptake during 313 continuous darkness. pC values were low and more or less constant from day 1 through to day 7 (p=0.487). However, N 314 uptake increased slightly under dark conditions. As already mentioned, Elphidia species possess chloroplasts 315 (kleptoplasts), which they incorporate from their food sources into their own metabolic cycle (Correia and Lee, 2000).

316
This aspect could be an important contribution to explain the light regime effects on food uptake rates. There are two 317 different explanations.

318
First, in complete darkness foraminifera could stop foraging and start feeding on their `own´ chloroplasts. Past 319 investigations showed that chloroplasts in Elphidium were exclusively derived from diatoms, making diatoms their preferred food source (Pillet et al., 2011). Our experiments showed that Elphidia had a significantly higher food uptake 321 after 7 days of starvation compared to the days before (Fig. 3). During the first 5 days, foraminifera may have either 322 stagnated with a reduced metabolism or they may have begun to digest their chloroplasts. For further investigations it 323 would be interesting to detect chlorophyll in foraminifera spectroscopically, since this molecule is found exclusively in 324 chloro-or kleptoplasts (Cevasco et al., 2015;Krause and Weis, 1991;Mackinney, 1941). One aspect to be discussed here 325 is the life time of (viable) kleptoplasts in foraminifera under natural conditions. For example, Nonionella labradorica 326 showed a strong seasonal variation in plastid viability (Cedhagen, 1991). According to Cedhagen (1991) (Fig. 2). After prolonged starving periods (>7d) in the dark, a starvation effect of this species 352 is noticeable (Fig. 3). The triggers for this effect are currently unknown. According to Jauffrais et al.  357 effect on C uptake, with higher C uptake from the diatom food. The effect of food type was even more pronounced for N 358 uptake, with clearly higher incorporation rates of N from the green algal food (see Tab. 3). However, different salinity levels caused significant differences with time. Since E. excavatum is one of the dominant species in the Kiel fjord 360 (Schönfeld and Numberger, 2007) and thus plays an important role in the turnover of organic matter, this aspect is 361 discussed in more detail here.

372
The Baltic Sea is the largest brackish water basin in the world (Voipio, 1981). During the sampling, the salinity was close 373 to 21 PSU (surface water). This brackish milieu leads to a low diversity of foraminifera (Hermelin, 1987;Murray, 2006).

374
According to Lutze (1965) benthic foraminifera of this region require a minimum of 11-12 PSU to survive. The lowest 375 salinity in our experiment was set slightly above this limit, with 15 PSU. Interestingly, the amount of incorporated N was 376 higher after 7 days at 15 PSU than at 25 PSU, and both pN and pC were highest at 20 PSU (considering mean values of 377 the uptake). Low salinities or strong salinity fluctuations can lead to smaller test sizes or test abnormalities of foraminifera 378 (Brodniewicz, 1965;Polovodova and Schönfeld, 2008). Only foraminifera without test abnormalities were taken for 379 experiments. After the feeding experiments, no visual influence of salinity on test abnormalities or new chambers were 380 recorded, but the time intervals in this study was likely too short for such observations. The influence of salinity on the 381 test structure of Elphidium in the Baltic Sea has already been investigated (e.g., Binczewska

399
In summary, we found significant differences in food uptake at different salinities. Elphidium excavatum seems to cope 400 better with lower salinities, which correlates very well with the brackish milieu in the Kiel fjord. An increase of the salinity 401 from 20 to 25 PSU caused more stress for the species than a reduction from 20 to 15 PSU (see reduced uptake of C and 402 N after 7 days at higher salinities in fig. 1). This once again demonstrates the good adaptation of E. excavatum to habitats 403 of lower salinity. Foraminifera can convert up to 15 % of the total annual flux of particulate organic matter in the Kiel 404 fjord (Altenbach, 1985). In addition, this region is strongly affected by eutrophication, making the Kiel fjord an interesting