Permanent ectoplasmic structures in deep-sea Cibicides / oides taxa – 1 long-term observations at in situ pressure 2 3

Deep-sea Cibicidoides pachyderma (forma mundulus) and related Cibicidoides spp. were cultured at in situ pressure 8 for 1-2 days, or 6 weeks to 3 months. During that period, fluorescence analyses following BCECF-AM (2’,7’-bis(29 carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester) or Calcein AM (4,5-Bis((N,N10 bis(carboxymethy)amino)methyl)fluorescein acetoxymethylester) labelling, revealed a persisting cytoplasmic sheet or 11 envelope surrounding the Cibicidoides tests. Thus, the Cibicidoides shell can be considered rather as an internal than an 12 external cell structure. A couple of days to a week after being transferred into high-pressure aquaria and adjusted to a pressure 13 of 115 bar, the foraminifera changed from a mobile to a more or less sessile living mode. During this quasi sessile way of life, 14 a series of comparably thick static ectoplasmic structures developed that were not resorbed or remodelled but, except for 15 occasional further growth, remained unchanged throughout the experiments. Three different types of these ‘permanent 16 structures’ were observed: A) Ectoplasmic ‘roots’ were common in adult C. pachyderma, C. lobatulus and C. wuellerstorfi 17 specimens. In our experiments single ectoplasmic ‘roots’ grew to maximum 700 times the individuals shell diameter and were 18 presumably used to anchor the specimen in an environment with strong currents. B) Ectoplasmic ‘trees’ describe rigid 19 ectoplasmic structures directed into the aquarium’s water body and were used by the foraminifera to climb up and down these 20 ectoplasmic structures. Ectoplasmic ‘trees’ were so far only observed in C. pachyderma and enabled the ‘tree’-forming 21 https://doi.org/10.5194/bg-2021-62 Preprint. Discussion started: 12 April 2021 c © Author(s) 2021. CC BY 4.0 License.

= 2) also abandoned ectoplasmic envelopes were observed, supporting the idea that the cytoplasmic envelope serves as matrix 140   directing into the water column. All static ectoplasmic structures may have shown continued growth but otherwise changed 162 little over the 3 months of observation. In one case braided ectoplasmic 'roots'even persisted after the termination of the 163 experiment when the two involved specimens were rinsed in deionized water and dried (Fig. 5g). We never observed that these 164 structures were in whole or in part resorbed. 165

Ectoplasmic 'roots' 166
The most frequent static ectoplasmic structures were 'root-like', extending along the bottom or adhering to the window of the

Ectoplasmic 'twigs' and pseudopodial network 234
Thick ectoplasmic structures extending into the water were termed ectoplasmic 'twigs' if the shape and position with respect 235 to the test remained essentially permanent during the experiment (Figs. 7-8). However, ectoplasmic 'twigs' are the least static 236 of the three described ectoplasmic structures and were only observed in C. pachyderma specimens so far. The first ectoplasmic 237 'twigs' appeared 3 days after transfer of C. pachyderma specimens into the aquaria (Fig. 7a). Additional structures were 238 eventually added over time (Fig. 7a-b), but the original structure was usually not modified (Figs. 7-8). Provided with the same 239 short and obviously adhesive side branches as ectoplasmic 'trees' (Fig. 6), the ectoplasmic 'twigs' probably support a more 240 delicate pseudopodial network (Figs. 7-8). In our experiment, C. pachyderma specimens exhibited a strong rheotaxis. In this 241 context it was observed that a specimen had positioned itself at the hole of the filter ring (where the food entered the aquarium).
twenty times the specimen's test size. Hereby, both the pseudopodial network and the respective supportive ectoplasmic 'twigs' 246 obviously allowed the animal to collect food from the water current (Figs. 8-10). When we shut down the pumps and, thus, 247 the current activity for some minutes (on May 26, 2017, 25 hours after feeding), the pseudopodial network, visualized by 248 adhering algae, collapsed (Fig. 8b), whereas the ectoplasmic 'twigs' kept their original shape (Fig. 8). The shape of the 249 specimen's ectoplasmic 'twigs' was neither affected by the presence or absence of the current nor by the speed of it (~0.1-5 250 cm/min (Wollenburg et al., 2018)). 251 For the specimen positioned at the hole in the filter ring, the development and extension of pseudopodia directing into the 252 water current during feeding was immediate (Fig. 10), however, the transport of collected algae towards the shell was extremely 253 slow. Seven hours after feeding, algae were still sticking to the pseudopodia and ectoplasmic 'twigs' and no or only low 254 amounts of fresh algae had reached the shell interior (Fig. 10f). Slow food ingestion was also reflected by the extremely slow 255 propagation of anastomoses over time. An anastomosis propagated less than 150 µm within 24 hours (Fig. 10). During and 256 following feeding, the number of granules in the ectoplasmic envelope, the ectoplasmic 'twigs', and pseudopodia were 257 significantly increased.

Torn ectoplasmic remains 295
When Cibicidoides specimens that were virtually sessile for weeks changed position, their static ectoplasmic structures could 296 obviously not be resorbed. These structures were either pulled along by the specimens, as shown for the ectoplasmic 'roots' in 297 Fig. 5, or torn off. Over the duration of the experiment, numerous ectoplasmic 'roots' and 'twigs', or what is supposed to be 298 parts of such structures, were flushed to the aquarium's window (Fig. 11). We had to increase the current speed through the 299 aquaria sporadically to get rid of the torn biomass and clear the view.