Mesocosm and Microcosm Experiments on the Feeding of Temperate Salt Marsh Foraminifera

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Mesocosm and Microcosm Experiments On the Feeding of Temperate Salt Marsh Foraminifera

Article in The Journal of Foraminiferal Research · July 2019 DOI: 10.2113/gsjfr.49.3.259

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MESOCOSM AND MICROCOSM EXPERIMENTS ON THE FEEDING OF TEMPERATE SALT MARSH FORAMINIFERA

Jennifer L. Frail-Gauthier1,*, Peta J. Mudie1, Alastair G. B. Simpson2 and David B. Scott1

ABSTRACT logic history since the Cambrian, because they are important biostratigraphic tools and key proxies for interpreting the Agglutinated foraminifera dominate in temperate salt paleoecology of ancient seas and fluctuations in relative sea marsh sediment, making them key indicators for monitoring level (RSL). The ecology of modern foraminifera has been sea level and environmental changes. Little is known about discussed in several major texts (Lee & Anderson, 1991; the biology of these benthic foraminifera because of difficul- Murray, 2006), but these reveal that many basic questions ties in distinguishing live from dead specimens in laboratory about feeding, growth, and reproduction remain unresolved cultures. We present data from 10 years of laboratory exper- and little explored since the early work of Bradshaw (1957, iments using comparisons of the agglutinant trochamminids 1968) and Arnold (1974). Limited information is also Trochammina inflata and Entzia macrescens and the mili- available in Murray & Alve (1999). This lack of knowledge olid Miliammina fusca with the calcareous rotalids Helenina is particularly acute for agglutinated marsh species and anderseni and Elphidium williamsoni. Specimens were taken other benthic foraminifera (Kitazato & Bernhard, 2014). from a laboratory mesocosm representing Chezzetcook Inlet, In effect, most environmental interpretations are based a cool-temperate salt marsh in eastern Canada. We deter- simply on relating generalised modern distributions to mined culture requirements for the agglutinated foraminifera abiotic conditions such as salinity, temperature, oxygen, and in Petri dishes over 10–12 week periods. Five inexpensive, marsh elevation. These must remain circumstantial until the non-terminal ways of identifying live organisms were devel- biological factors that constrain foraminiferal occurrences oped: spatial movement, detritus-gathering, attachment, clus- are understood, including their complex feeding habits tering, and test opacity. Comparison with rose Bengal staining (Goldstein, 1999; Mojtahid et al., 2011). For example, showed <10% diversion for calcareous species and T. inflata the notoriously high patchiness of foraminifera (e.g., Lee, but M. fusca was over-counted by >30%. Terminal chambers 1974) could be governed by food availability, feeding meth- of Trochammina inflata were examined by transmission elec- ods, competition with meiofauna, abiotic factors (salinity, tron microscopy to visualise food consumption and identify elevation), or a combination of some or all of these. food in digestive vacuoles, both in specimens from mesocosm A key objective of our study is to expand knowledge of and in culture. Bacteria and unidentified detritus in the vac- the trophic niches that control the spatial and temporal pop- uoles establish that this agglutinated species is a saprophagous ulation dynamics of temperate salt marsh foraminifera evi- and bacterivorous detritivore. The adhesive secretions by these dent in pioneering Quaternary paleo-sea level studies (e.g., species apparently help them gather and possibly farm food Scott et al., 2001 and references therein; Kemp et al., 2011, while being relatively immobile in the sediments. Our obser- 2013). Within the suite of salt marsh marker species, aggluti- vations of movement and feeding orientation in the agglutinated taxa, such as Entzia (Jadammina) macrescens (Brady, nants suggest links between form and function that underscore 1970), Miliammina fusca (Brady), and Trochammina inflata their value as ultra high resolution sea-level proxies. Meso- (Montagu), are the main tools for paleoenvironmental work cosm biomass and abundance counts show that foraminifera in coastal environments because of their better preserva- represent >50% of the meiofaunal biomass, emphasising their tion potential in acidic salt marsh sediments compared to importance in the food web and energy-flow dynamics of tem- calcareous taxa (reviewed in Berkeley et al., 2007). Most perate salt marsh systems. trophic niche studies, however, have focused on calcareous taxa common on mudflats or in deeper coastal water, in- INTRODUCTION cluding Ammonia beccari/tepida (Cushman), Haynesina germanica (Ehrenberg), and Elphidium spp. Montfort (Dupuy Although foraminiferal biology has been extensively et al., 2010; Seuront & Bouchet, 2015; Jauffrais et al., 2016). studied for over 150 years (e.g., Myers, 1943; Arnold, 1974; Thus, previous conclusions about the behavioural, feeding, Loeblich & Tappan, 1988; Sen Gupta, 1999; Murray, 2006; and biotic interactions of salt marsh benthic foraminifera Kitazato & Bernhard, 2014), details of their feeding remain are based on a restricted part of the total assemblage and enigmatic. Most studies of foraminifera focus on taxonomy, do not consider the agglutinated species that are widespread faunal assemblages, or distributions in marine and brackish throughout the entire salt marsh and often present in ex- environments, both around the world and throughout geo- tremely high abundances. Direct observation of feeding and behavioural habits in dominant salt marsh foraminifera is crucial for inferring 1 Department of Earth Sciences, Life Science Centre, Dalhousie Uni- their role in the ecosystem, but few studies have exam- versity, 1355 Oxford Street, Rm 3006, Halifax, Nova Scotia, Canada ined these species in incubator (microcosm) cultures (Gold- B3H 4R2 stein & Alve, 2011; Weinmann & Goldstein, 2016; van Dijk 2 Department of Biology, Life Science Centre, Dalhousie University, 1355 Oxford Street, P.O. Box 15000, Halifax, Nova Scotia, Canada B3H et al., 2017). Past observations and/or feeding experiments 4R2 have shown that benthic foraminifera can be photosyn- * Correspondence author. E-mail: [email protected] thetic (hosting diatoms), chemosynthetic (hosting bacteria),

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fluorescent dye (CellTrackerTM Green, CTG) is very expensive and also requires specialised fluorescence microscopes for time-consuming stain-reaction studies that are difficult to quantify (Figueira et al., 2012). We sought to determine suitable non-harmful and inexpensive methods for examining living populations of agglutinated foraminifera with thick, opaque tests by compiling a check-list of criteria to distinguish living benthic salt marsh foraminifera from dead specimens in laboratory experiments. The salt marsh mesocosm in the Dalhousie Aquatron (Scott et al., 2014) allowed implementation of feeding trials with foraminifera and meiofauna from a cool temperate environment on a year-round basis over a period of five years. Additionally, new observations could be made on the key agglutinated and calcareous salt marsh paleo-sea level proxies that have been overlooked in past biological studies of foraminifera and marshes in warmer regions (e.g., Texas and California: Bradshaw, 1957, 1968; Georgia and Florida, USA: Weinmann & Goldstein, 2016). Our work as- Figure 1. Five species used for culturing and feeding experiments. sesses the feeding habits of the mesocosm temperate climate Top row: agglutinants (A) Miliammina fusca, with small horse-shoe- salt marsh foraminifera by direct observation of specimens shaped terminal aperture; (B) Entzia macrescens, with arenaceous test in vitro and by investigation of detritus accumulation around and small, round terminal aperture; (C) Trochammina inflata, organics tests. The accumulation of detritus at the terminal cham- at aperture (arrow). Bottom row: calcareous (D) Elphidium williamsoni and (E) Helenina anderseni. Scale bar = 100 µm. ber aperture (Goldstein, 1999) or around the whole specimen (Arnold, 1974) has been previously called formation of “feeding cysts” (Heinz et al., 2005). Transmission electron herbivorous (grazing on diatom mats), detritivorous, carniv- microscopy (TEM) of the food vacuoles in the agglutinant orous, or parasitic (e.g., Lee & Anderson, 1991; Bernhard & Trochammina inflata was used to provide novel information Bowser, 1992; Sen Gupta, 1999; Suhr et al., 2003; Mojtahid on feeding mechanisms in selected individuals fed with un- et al., 2011; Jauffrais et al., 2016). Their crucial importance altered marsh mud or with marsh bacteria isolates. Biomass in some ecosystems is evident from the fact that benthic was also calculated to show how much benthic foraminifera foraminifera in deep waters in the Arctic (Sea of Okhotsk) contribute to the organic matter (OM) and carbon budgets and on mudflats along the North Sea coast constitute over of the salt marsh sediment. 50% of meiofaunal (63–500 μm) biomass and abundance in In summary, the objectives of this study are three-fold: small-scale (<5cm3) patches; their production values range 1) to identify low-maintenance, non-terminal methods of − − from 90 to >5000 mg C m 2 yr 1 (Chandler, 1989). distinguishing life status for three agglutinated and two The primary objective of the work reported here is to calcareous salt marsh foraminifera in a laboratory culture examine the feeding and behaviour of the dominant salt setting; 2) to determine feeding modes in Trochammina marsh taxa used in the classical RSL studies of Scott & inflata through TEM examination; 3) to determine spe- Medioli (1980). The selected species are (Fig. 1): agglu- cific feeding habits of salt marsh foraminifera as well as tinants Trochammina inflata and Entzia macrescens in their biomass. This set of observations will contribute to Class Globothalamea (Pawlowski, Holzmann & Tyszka, new understandings of agglutinated and key calcareous 2013), Order Lituolida (Ben Gupta, 1999); agglutinant salt marsh foraminiferal responses within their ecological Miliammina fusca in Class Tubothalamea (Pawlowski, niches, how foraminifera might respond to environmental J., Holzmann, M., Tyszka, J., 2013), Order Miliolida changes affecting their food sources, and how their biomass (Delage & Hérouard, 1896); and calcareous species El- contributes to carbon budgets and sediment energy fluxes. phidium williamsoni [Haynes, 1973] and Helenina anderseni [Warren, 1957]) in Class Globothalamea, Order Rotaliida (Loeblich, A. R., Tappan, H., 1987). To perform these METHODS studies, it was necessary to develop new methods for non- Sampling, Foraminiferal Supply, and Live harmful examination of opaque-walled agglutinated benthic Observations foraminifera to distinguish living from dead specimens. Six distinguishing criteria are described in Arnold (1974), Nearly 500 total surface sediment samples with living but these were based on thin-walled calcareous taxa with foraminifera and potential food came from the mesocosm translucent walls. Most previous studies that have inves- marsh developed in the Aquatron facility at Dalhousie Uni- tigated ways to distinguish between living and dead cells versity or from in situ surface sediments from Chezzetcook in assemblages of foraminifera in surface sediments have Inlet, Nova Scotia from 2007–2017. The size of samples re- relied on fixing (i.e., killing) the specimens prior to staining moved for cultures varied from 2 to 10 cm3,withsurface (with rose Bengal or Sudan Black) or examining specimens scraping less than 0.5 cm thick. The samples were taken by transmission electron microscopy (TEM) or scanning from the mesocosm marsh elevational zone likely to pro- electron microscopy (SEM). A non-terminal method using vide maximum numbers of the selected foraminifera needed

for detailed study, based on their known relative abundances of marsh water and sediment were taken from the meso- (Frail-Gauthier, 2018, table 2.4). cosm with pipettes and placed in Petri dishes. A sterile inoc- Samples were gently washed through nested 500-µmand ulating loop was used to transfer 0.1 ml of samples to agar 63-µm sieves (Fig. 2A, i–iv) using filtered seawater adjusted plates, streaking over the entire plate. Two plates were pre- with distilled water to keep salinity at 15 psu. The 500-µm pared for each sample of marsh water or sediment, totaling mesh removed large debris and separated foraminifera from eight plates. They were covered and placed upside down in consolidated organics and sediments. The 63–500 µmsed- a sterile plastic box for four days at room temperature. Af- iment fraction was poured into Petri dishes (10 cm in di- ter five days, five individual bacterial colonies that were visi- ameter) and 2-µm filtered sea water + distilled water was bly different (colony colours and shapes) were removed and added to match the average salinity of the corresponding salt streaked onto new TSA plates. These five plates were covered marsh zone measured in the field. Petri dishes were covered and stored upside down for three days, then examined daily to minimise evaporation. Dishes were left at room tempera- for five days under a dissecting microscope to confirm pu- ture (∼22°C). rity. These five cultures were transferred to fresh TSA plates Dishes were examined under a stereomicroscope (10–50x weekly for the first month after isolation, and subsequently total magnification). Living individuals in the size range of once a month to maintain the cultures. These bacterial cul- about 150–300 µm, generally recognised by movement or tures were used as food sources both for general feeding ex- chambers containing cytoplasm, were transferred by fine periments and for some TEM observations. brush or pipette to a smaller Petri dish (5 cm in diameter; Fig. 2A, v–vi). The smaller dishes contained a small amount Foraminiferal Feeding Observations of water (up to a few millimeters deep) with salinity adjusted as above. The dishes were left for 24 hours, and foraminifera General Methods were reevaluated the following day to remove dead individ- Feeding culture experiments were performed using the five uals that were previously thought to be living. For dishes μ dominant species of foraminifera found at Chezzetcook In- containing calcareous species, 0.2 l of calcium (Hagen® let. These species included three agglutinated species (Mil- Fluval® Sea Calcium) and alkaline (Hagen® Fluval® Sea iammina fusca, Entzia macrescens,andTrochammina inflata) Alkalinity) solutions were added to give sufficient calcium and two calcareous species (Helenina anderseni and Elphid- carbonate for test maintenance, growth, and/or sexual or ium williamsoni; Fig. 1). We note that M. fusca is proba- asexual reproduction. All dishes were left at ambient room ∼ ° bly derived from calcareous ancestors (Habura et al., 2006). temperature ( 22 C) and light. In general, the length of time Most of our experiments used T. inflata because of the par- for the individual experiments ranged from days to weeks ticular need for data on agglutinated species and because of with observations made at daily intervals (average time of its relatively large size and conspicuous motility. four weeks). Some details for individual studies, such as To prepare cultures for feeding experiments, live Culturing Experiments, are given in other Methods sections foraminifera were taken from the stock culture dishes below. (Fig. 2A, v–vi) after removing any attached detritus with a fine brush. Five individuals of the same species were Preparation of Food placed in a small Petri dish (5 cm in diameter) with ∼5ml of filtered seawater (2 µm) adjusted with distilled water to Because many benthic agglutinated foraminifera are as- correspond to average field conditions (30 psu for calcare- sumed to be detrital and bacterial feeders, the stocks of food ous species and 15–20 psu for agglutinated species); the used for maintenance and experimentation were 1) unaltered resulting water layer was 4 mm deep. The dishes were kept mesocosm salt marsh mud; 2) filtered detritus; and 3) cul- at room temperature (22°C) with natural ambient light from tured salt marsh bacteria. For the first “field” mud food windows and overhead fluorescent lights (from 8 am to source, a small amount of salt marsh sediment was scraped 5 pm). If the foraminifera were being fed bacteria, the salt from the mesocosm high-salinity middle marsh (20 psu) and water was further filtered through a 0.45-µm syringe filter placed in a dish with ambient sea water from the mesocosm. immediately before being added to the dish. Food types Sediment was stirred to separate large particles and disperse used in various feeding observation experiments included 1) the material. One or two pipette drops of this stirred mud control (no added food; for meiofaunal feeding studies); 2) were then added to culture dishes containing foraminifera. two drops of marsh mud + water (for general foraminifera Secondly, to prepare filtered detritus, mesocosm marsh 3 feeding observations); 3) two drops of filtered detritus (for sediment samples (2 cm ) were washed over stacked 45- a 12-week culture experiment); and 4) a loop (∼10 µl) of µ µ μ m and 63- m sieves. The remnant 45–63 m detrital food cultured marsh bacteria (for one TEM experiment). Dishes material contained no meiofauna, macrofauna, and few were examined every 1–2 days for activity, and water levels foraminifera but included particulate and dissolved organic and salinity were adjusted and fresh food was added weekly matter, bacteria, and non-filamentous micro-algae. The de- for experiments longer than one week. trital food source was maintained in a loosely-capped vial with filtered salinity-adjusted seawater (as above) and re- Bacteria-fed Trials for TEM Observations plenished on a weekly basis during the experiments. Thirdly, for cultured bacteria, new and/or sterile equip- Five living T. inflata were placed in five separate Petri ment was used, work stations were thoroughly sterilized, and dishes (5 cm in diameter; Fig. 2A, vi), one dish for each of transfers occurred in a fume hood. All bacteria were grown five isolates (labelled 1 through 5) of bacteria. Foraminiferal on Tryptic Soy Agar (TSA) plates. Small (<1 ml) samples specimens were left overnight to allow feeding vacuoles to

Figure 2. Methods of sampling protocols and Transmission Electron Microscopy preparation. (A) Samples (ii) were scraped from mesocosm marsh surface (i), washed through stacked sieves (iii), with the sieved residue stored in 15 psu 10-cm Petri dishes (iv). Living foraminifera were picked from dishes (v) and placed in separate 5-cm dishes per species (vi) to explore a variety of feeding conditions (as explained in the text) and used for a variety of experiments, including TEM (vii) and stain reactions. (B) Schematic of polymerising (i, ii), thin-sectioning (iii, iv, v), and mounting (vi) of Trochammina inﬂata sections for examination with TEM (vii).

empty, then placed in new dishes with 20 µl of one of the section although it was attempted by Bender & Hemleben five bacterial cultures, and left for two days. Overall feed- (1988, pl. 2, fig. 5). The TEM sections of the wall of Mil- ing observations were made by examining dishes under the iammina fusca were studied by Bender & Hemleben (1988, stereomicroscope before preparing them for TEM study of pl. 1, figs. 1–5) and Habura et al. (2006, figs. 5–7). To the chamber contents. examine “field-fed” foraminifera, live Trochammina inflata specimens were processed immediately after taking samples from the mesocosm middle marsh (Fig. 2A, i), and six spec- Transmission Electron Microscopy of imens were placed in each of two Eppendorf tubes (Fig. 2A, Trochammina Inflata vii). Following the method of Bender & Hembleben (1988), Trochammina inflata was chosen for TEM work because the foraminifera were then fixed with 25% glutaraldehyde in of its large, round chambers compared to the narrow a buffer of 0.05M sodium cacodylate and 5% w/v sucrose for chambers of Entzia macresens, which make it difficult to 90 min, rinsed three times with buffer, post-fixed for 90 min

Figure 3. Stereomicroscope photo-image of living salt marsh foraminifera. (A) Three clumped species: Helenina anderseni (i), detritus-covered Trochammina inflata (ii), and Miliammina fusca (iii); (B) clumped M. fusca (top) and T. inflata, with detritus around terminal aperture; (C) Entzia macrescens with detritus concentrated around aperture; (D, E) Helenina anderseni covered in gathered detritus; (F, G) living Elphidium williamsoni with alga-filled yellow (F) or green (G) cytoplasm in all but terminal chambers. Scale bars = 100 μm.

in 1% OsO4 in the same buffer, and finally rinsed six times with distilled water. The tests of the fixed foraminifera were punctured with a fine needle to make at least two holes for Figure 4. Organic linings of foraminifera from mesocosm middle penetration of resin into the cytoplasm. The punctured marsh. (A) Mid-focus showing mostly cytoplasm-filled chambers; (B) specimens were then dehydrated though a series of aqueous high-focus showing remnant external organic membrane, possibly part ethanol mixtures (30%, 50%, 70%, 90%, 95%), then 100% of a sticky mucous-like feeding network that assists in detritus gathering; (C, D) Two dead specimens of T. inflata showing the organic lining ethanol, followed by two Spurr’s resin ethanol mixtures structure and differences in micropore-structures in the chamber walls (30%, 60% resin), and finally five 100% resin changes over between terminal and inner chambers; (E, F) M. fusca with stained cy- >48 hours. The infiltrated foraminifera were embedded toplasm in chambers surrounded by a yellowish organic layer. Scale bar in well molds containing partially polymerised resin to in (A) applies for all images. prevent their sinking, then fully polymerised at 60ºC(Fig. 2B, i–ii). Embedded foraminifera were sectioned (100 nm thickness) using a Leica EM UC6 ultramicrotome with a Miliammina fusca, Helenina anderseni;90microcosmstotal). diamond knife (Fig. 2B, iii–v). The outermost chamber Each microcosm (Fig. 2A, vi) contained 10 living individuals with cytoplasm (terminal or penultimate chamber) was in 5 ml of filtered seawater (20 psu, 22ºC) to which a drop of sectioned to examine the intra-shell cytoplasm closest to filtered detritus was added weekly, along with adjustments in the apertural pseudopodia used in food collection. Sec- water to account for evaporation. At the end of the experi- tions were stained with uranyl acetate and lead citrate and ment, we made a comparison of our observations on vitality examined using a FEI Tecnai-12 transmission electron vs. mortality using the traditional rose Bengal methods as microscope (Fig. 2B, vi–vii). Interpretations of TEM photo- explained below. images were validated by Susan Goldstein (University of Every week, each dish was examined under a stereomi- Georgia) and Emmanuelle Geslin (Université Angers) as croscope (10–50x magnification) and all living foraminifera well as by comparison with images from Anderson & Lee were counted; apparently dead individuals were removed. (1991), Goldstein & Corliss (1994), and LeKieffre et al. At the end of the 12 weeks, all samples were stained with (2018). 2–3 drops of rose Bengal, and 0.5 ml of ethanol was added to each dish to confirm the assessment of live condition (af- Culturing Experiments to Validate Live Specimens ter 24 hours, foraminifera with living protoplasm at the time of application should be stained a bright rose pink in major- To determine the best way to recognise live foraminifera ity of the test chambers, not simply the terminal chambers; without use of stains or expensive fluorogenic probes, Petri Walton 1952). A longer waiting period was not used because dish cultures of foraminifera were maintained for 12 weeks; it allows for microbial degradation and further staining un- cells were examined for feeding activity or movement and related to the vitality of the foraminifera. The final count of counted weekly to determine changes in population num- pink specimens was used to assess that all foraminifera re- bers. Thirty Petri dish microcosms (5 cm in diameter) were maining at the end of the feeding study were correctly clas- examined for each of three species (Trochammina inflata, sified as living.

Figure 5. Sections of direct-from-mesocosm Trochammina inﬂata. (A) Degraded bacteria (B) in a food vacuole (V); (B) Food vacuole (V) including possible bacteria in various stages of degradation (DB). Mitochondria (M), probable nucleus (N), ﬁbrillar vesicles (FV), and putative peroxisomes (P) are also visible.

Feeding Trials with Associated Invertebrate selected for these experiments. Cultures were examined with Meiofauna a 10x stereomicroscope after 24 hours and 48 hours, including counts of individuals and notes on interactions between To examine potential invertebrate meiofauna-forami- them, such as direct feeding or items in guts. nifera interactions, separate cultures were made that used foraminifera combined with meiofauna or small macrofaunal species (gastropods, polychaetes, oligochaetes, ostracods, soil mites). Petri dishes were prepared from mesocosm Biomass and Abundance Calculations: Foraminifera samples, and foraminifera and meiofauna were combined as and Meiofauna shown in Table 1. Samples for this experiment were taken For background information on the biomass of from selected elevational zones of the marsh mesocosm and foraminifera and associated meiofauna (defined here as the variable numbers per dish reflect the sample patchiness. animals of size 63–500 μm) within the sediment community The scope of this small-scale experiment did not permit tax- of salt marshes, multiple 2.5-ml samples of salt marsh mud onomic definition to species level except when there were two from Chezzetcook Inlet and each of the four high-salinity kinds of foraminifera (Trochammina inflata and Miliammina mesocosm zones were washed over 63–500 μm sieves. The fusca) in one dish; the most abundant “predator” (carnivore, sievedsampleswerefixedwithethanolandstainedwith omnivore, deposit feeder) in each taxonomic category was 2 ml rose Bengal solution, then re-washed over a 63-μm sieve to remove residual rose Bengal and ethanol after 24 hours. Individuals were counted and sorted into major Table 1. Groups of feeding trials with paired foraminifera and meio- taxonomic groups: foraminifera, nematodes, polychaetes, fauna/small macrofauna or paired foraminifera used in the competitive oligochaetes, ostracods, copepods, amphipods, isopods, feeding experiments. At the start, each Petri dish contained foraminifera and a meiofaunal group (13 dishes with oligochaetes, 3 with poly- fly larvae, and mites. The wet and dry weights were then chaetes, 2 with gastropods, 4 with ostracods, 4 with soil mites). After recorded using a Sartorius Analytic microbalance (precision 48 hours (end of experiment), observed changes are shown in parenthe- ±0.01 mg) for each group of taxa within each sample. The ses (number of individuals remaining). wet weights were measured after evaporation of excess water; dry weights were obtained after drying in a Boekol # Oligochaetes # Foraminifera # Polychaetes # Foraminifera Model #1078 drying oven at 65ºC overnight. 15(3)15 3 515 2 315 Data Analysis 1 5 # Ostracods # Foraminifera 1 535Results from feeding trials, culturing experiments, and 15 (14) 10 (8) 1 3 5 5 (4) 3 (2) 3 TEM studies are presented as qualitative observations. 55(4)15Quantitative analyses (descriptive mean and standard devi- 5 1 # Soil mites # Foraminifera ation statistics) compared the in-culture living counts to the 21011final counts of foraminifera stained with rose Bengal. Meio- 1515(4)faunal feeding trial results are shown in Table 1. Biomass 15110 1 515(mg) and abundance calculations are based on the mean and # Gastropods # Foraminifera # T. inflata # M. fusca standard deviations of the n samples per zone. Percents of 10 8 10 10 biomass and abundance are based on the total of meiofauna 11 plus foraminifera.

Figure 6. Food vacuoles from Trochammina inflata fed with Bacterium 4. (A) Vacuole showing many bacteria (B). (M)–mitochondria; (P)– peroxisomes; (FV)–fibrillar vesicles. Enlarged images show a non-degraded bacterium in cross-section (B*) and partially degraded bacterium (DB*). (C) Several food vacuoles (V), including one with undigested bacterium. Enlarged images (D, E) show details of the vacuole contents and the bacterium, also other bacteria in partially digested state [bottom right vacuole in (D)]. Note also mitochondria (M), peroxisomes (P) and lipid droplets (L). Note different scale bars.

RESULTS the aperture-side ventrally and pulling the test along the Petri dish with extended pseudopodia at a unidirectional General Feeding Observations and Identifying Living −1 Specimens speed of up to 2 mm hr . After one day, individuals were often up the sides of the 1-cm tall Petri dish or on the oppo- Of the agglutinated salt marsh foraminifera in this study, site side (50 mm). No lateral movement was ever observed Trochammina inflata was the only species seen with an ex- directly in Miliammina fusca or Entzia macrescens but af- tended pseudopodial (rhizopodial) network that provided ter 24-hr intervals, individuals of all three species were com- visible motility. Movement of living specimens could be ob- monly found congregated in pairs or small clusters as shown served with a stereomicroscope or inverted compound light for T. inflata, M. fusca,andHelenina anderseni in Figures microscope (LM). Trochammina inflata moved by orienting 3A and 3B, whether single or multiple species were present,

Figure 7. Intra-shell extracellular environment of T. inﬂata fed with bacteria (all are rod-shaped species). (A) Individual fed with Bacterium 1. (B) Individual fed with Bacterium 3, showing mostly extracellular material outside of the darker intracellular cytoplasm top left. (C) Individual fed with Bacterium 1 with the red square expanded in (D) to show details of bacteria. Note diﬀerent scale bars.

indicating that slow, cryptic movement occurred in the Detritus-free specimens had alga-filled cytoplasm in many less active species. Miliammina fusca also displayed cryptic chambers, except the terminal chamber. The cytoplasm movement in re-orienting its test from horizontal to vertical was orange-brown in H. anderseni and greenish-brown in (aperture-side down) position. E. williamsoni (Figs. 3E–G). Test and organic linings of ag- Regardless of motility, a good indicator of living spec- glutinated species, especially M. fusca, were less transparent imens with extended pseudopodia/cytoplasm was the an- than the calcareous species. However, their cytoplasm-filled choring of the foraminifera to the bottom of the Petri dish. inner chambers were consistently darker than tests without A gentle swirl of the dish distinguished those attached to the cytoplasm when viewed either under inverted light or direct bottom (living) from those that floated freely (dead). An- light. The three agglutinated species had viscous, opaque other common indicator of live specimens was the presence white cytoplasm regardless of food source. Some living spec- of detritus at the aperture (Figs. 1C, 3C) or covering the in- imens that were damaged when moved by brush or pick dividual. After adding detritus to the living cultures, indi- showed this living cytoplasm oozing out of cracks or holes viduals covered themselves in sediment, becoming “feeding in the test. cysts” sensu Goldstein & Alve (2011) within 24 hours (Figs. Light microscopy images of corresponding organic lin- 3A, D, E). Miliammina fusca often oriented itself aperture- ings from the mesocosm foraminifera after removal of the side down (vertically) on the Petri dish, with a bolus of detri- test with acids (Frail-Gauthier & Mudie, 2014) show fur- tus around the aperture. Clean specimens of E. macrescens ther details of fine structure and sometimes organic exudate also became surrounded in a thin layer of detritus from outer chambers (Fig. 4). Other LM images (Mudie & (Fig. 3C) with most particles concentrated at its aperture. Yanko-Hombach, 2019) show that 5% acetic acid applied to When detritus-free agglutinated specimens of T. inflata were T. inflata from Chezzetcook Inlet salt marsh does not dis- fed bacteria cultures, thin, translucent whitish “clouds” solve the arenaceous test but loosens the fibrous organic ma- would surround the entire test of the living foraminifera trix to show the interconnection of fibre-coated silica grains, within 24 hours. as described by Bender & Hembleben (1988). The calcareous species Elphidium williamsoni and Helen- ina anderseni have thin, shiny white, translucent tests, and Transmission Electron Microscopy they were the easiest of the experimental taxa to distinguish as being alive because of visible changes in cyto- Trochammina inflata specimens taken directly from the plasm contents. In large dishes of unwashed, unsieved mud, mesocosm contained rounded food vacuoles up to 5 μm both species would be found attached to filamentous algae. across lying within regions of cytoplasm that also included

Table 2. Counts of observed living foraminifera after a 12-week culturing experiment compared to counts after staining with rose Bengal. In the “Count error” and “Average diﬀerence” columns, a negative number means fewer foraminifera were stained with rose Bengal than originally counted alive in culture; a positive number means that the stained count gave more living foraminifera than originally counted alive in culture. SD = standard deviation.

Live count Rose Bengal Species (original count) stained count Count error per dish Average diﬀerence Conclusion

Helenina anderseni 130 total (Mean 4.48 131 total (Mean 4.52 Range −1to+3; SD +2.97% SD 0.31 No meaningful net per dish; SD 1.98) per dish; SD 1.88) +0.03 diﬀerence Trochammina inﬂata 141 total (Mean 4.7 128 total (Mean 4.27 Range −2to0;SD−0.43 −8.27% SD 0.13 Rose Bengal shows per dish; SD 1.78) per dish; SD 1.6) fewer living than counted. Miliammina fusca 182 total (Mean 6.1 123 total (Mean 4.1 Range −6to+1; SD −33.9% SD 0.35 Rose Bengal shows per dish; SD 1.66) per dish; SD 2.48) −1.97 far fewer living than counted.

typical cell contents such as mitochondria, putative peroxi- Miliammina fusca, living counts based on culture observa- somes, and fibrillary vesicles (Fig. 5). A variety of items was tions (cytoplasm, activity, etc.) estimated more living indi- observed in the food vacuoles, including bacteria in various viduals than were stained by rose Bengal. Miliammina fusca stages of degradation and degrading cellular material of un- were the least successfully identified by culture observations, known origin (Fig. 5). overestimating living specimens by ∼34%comparedtothose Specimens fed with Bacterium type 4 had vacuoles with stained with rose Bengal. bacterial cells in various stages of degradation (Fig. 6). Bac- teria fed to the foraminifera were observed as a thin film outside of the test and also inside the test (intra-shell), external Meiofaunal Feeding Trials to cytoplasm (Fig. 7). The 48-hour feeding experiments with meiofauna produced more obscure results than the feeding trials with detri- Determining Living Foraminifera in Culture tus and bacteria. No direct feeding observations were noted in the meiofauna experiments, but 6 of 27 Petri dishes had No noticeable size increase was observed in the experi- missing foraminifera, oligochaete, or ostracod individuals ments reported here or in trial growth-culture experiments (Table 1). These results could indicate that some cannibal- done in 2005, so we did not attempt to use specimen growth istic or inter-species feeding took place although there was as a criterion to assess the vitality of the specimens in our no visible evidence of this inferred feeding during the times microcosms. of observation. There were also no identifiable foraminifera The key determinants for identifying the living arena- or meiofauna within the guts of remaining individuals in the ceous and calcareous foraminifera in our cultures, with- dishes, and no evidence of escaped individuals outside of the out disturbing or killing them, are: (1) presence of detri- dishes. tus or sediment balls sustained by streams of cytoplasm and/or pseudopodia; (2) detritus or sediment at the aperture opening; (3) adherence to the bottom/sides of the Biomass and Abundance Petri dish; (4) movement over a short period of time (<24 hours); (5) presence of visible pseudopodial networks seen Multiple 2.5-ml samples of 63–500 μm specimens were by stereomicroscopy; (6) “fuzzy” appearance because of counted from each zone of the mesocosm (Table 3). small amounts of cytoplasm extruded from micropores; (7) Foraminifera are the most abundant category of meiofaunal opaque appearance in contrast to translucent tests as seen organisms, and also contribute the most to the total meio- in inverted light; (8) orange-brown or greenish-brown-filled faunal biomass. In the mudflat sediment, foraminifera ac- tests of calcareous species; (9) congregation of individuals count for almost half of the meiofaunal abundance (49%) (Figs. 2, 3A, 3B). and almost one quarter of the meiofaunal biomass (24%). Rose Bengal was used to count the living foraminifera in The dominance of foraminifera increases throughout the cultures of T. inflata, M. fusca,andH. anderseni after 12- higher elevations of the marsh, exceeding an average of week incubations in which they were fed with filtered de- 75% in both abundance and biomass in the middle and tritus. Table 2 compares the numbers of live foraminifera high zones (Table 3). After foraminifera, nematodes dom- estimated using the motility/activity/attachment criteria re- inate meiofaunal abundance in the mudflat (20%) and ported above (‘Live Count’) to those determined by this low marsh (11%), and soil mites dominate in the mid- (post-mortem) rose Bengal staining. dle (14%) and high marsh zones (7.5%). Flatworms (39%) Overall, staining with rose Bengal gave correct estimates and oligochaetes (28%) have the highest biomasses after for living counts in calcareous individuals of Helenina an- foraminifera in the mudflat and low marsh, respectively. derseni, with 131 individuals being identified as living us- Soil mites have the highest biomass after foraminifera of ing rose Bengal and only a small (∼ 3%) overestimate (Ta- the meiofauna in the middle (9.3%) and high marsh zones ble 2). For agglutinated species of Trochammina inflata and (10%; Table 3).

Table 3. Mean abundance and biomass values for multiple 2.5 ml 63–500-μm sieved samples of marsh sediments from each zone (mudﬂat through high marsh) of the high-salinity (20–33 psu) mesocosm marsh. The number of replicates for each zone is given in parentheses for each zone. Standard deviation values are given as ±. Average percentages of foraminifera for each zone are shown in the bottom row.

Abundance Dry weight (mg) Abundance Dry weight (mg) Abundance Dry weight (mg) Abundance Dry weight (mg) Marsh Zone Mudﬂat (6) Mudﬂat (6) Low (7) Low (7) Mid (5) Mid (5) High (4) High (4)

Foraminifera 157.2 ± 67.4 3.8 ± 3.1 657.9 ± 194.4 12.8 ± 4.9 624 ± 216 6. 2 ± 2.5 477.5 ± 103.1 4.7 ± 2.1 Nematodes 64.3 ± 55.6 0.7 ± 0.4 96.1 ± 67.5 1.8 ± 3.6 29.4 ± 33.4 0.4 ± 0.3 20.8 ± 18.7 0.3 ± 0.5 Ostracods 42 ± 28.4 1.3 ± 1.5 5.9 ± 4.5 0.2 ± 0.2 1 0 Polychaetes 5 ± 6.7 0.3 ± 0.4 9 ± 6.9 0.2 ± 0.2 7.5 ± 5.4 0.3 ± 0.3 7.7 ± 40.2± 0.1 Oligochaetes 6 ± 4 0.03 ± 0.06 13.9 ± 8.2 6.2 ± 14.8 6.4 ± 4.6 0.06 ± 0.05 15.7 ± 9.3 0.2 ± 0.2 Copepods 23.2 ± 15.5 2.2 ± 3.4 62.4 ± 43.2 0.5 ± 0.4 9.8 ± 6.1 0.3 ± 0.5 13.7 ± 12.4 0.07 ± 0.1 Midge larvae 19.3 ± 15.6 0.97 ± 0.8 5.3 ± 4.4 0.3 ± 0.3 3 0.2 1 0 Soil mites 2 ± 10.4± 0.4 11.5 ± 4.3 0.2 ± 0.1 113 ± 50.9 0.8 ± 0.7 43.3 ± 29.6 0.6 ± 0.5 Flatworms 2 0 1 0 Amphipods 2 0 % Foraminifera 49% 23.7% 76% 57.6% 78.7% 75.8% 82.3% 76%

DISCUSSION The other marsh agglutinants displayed only cryptic movement, expressed as clumping and test re-orientation Non-Terminal Criteria Distinguishing Living over a period of 24 hours. The faster movement of the tex- Foraminifera tularid T. inflata may be related to the fact that the test There is extensive literature on criteria for distinguish- is wider across the umbilical-spiral plane compared to the ing calcareous living foraminifera using non-terminal meth- narrower test of E. macrescens (Figs. 1, 3, 4) or the oval- ods (Arnold, 1974; Bernhard, 2000) but only sparse infor- shaped tubothalamid M. fusca. Possibly, movement by pseu- mation on the agglutinated species that characterise eleva- dopodial traction is easier for benthic foraminifera with tional zones of salt marshes. The opportunity for sampling a discoidal test (Fig. 1C) than for those with an ovoid from a salt marsh mesocosm allowed weekly monitoring test (Figs. 1A, B). The much larger primary aperture in of feeding trials and examination of the feeding habits of T. inflata, compared to the small pore-like primary aperture two agglutinated and one calcareous RSL marker species in E. macrescens may also explain the faster movement of over a period of 12 weeks. This time-series feeding study de- the former species (see Loeblich & Tappan, 1988, T. inflata: pended on establishing inexpensive non-destructive methods pl. 129, figs. 20–23; E. macrescens: pl. 133, figs. 7–13). Slower to distinguish living from dead specimens of selected marker movement might also be expected for the ovoidal M. fusca species placed in microcosm cultures after removal from the with a small horseshoe-shaped primary aperture (Fig. 1A). marsh mesocosm. Quick and effective ways for selecting liv- Other non-salt marsh foraminifera, such as the infaunal ing foraminifera from stock samples was also important for rotalid Pseudorotalia (Rotalinoides) gaimardi (d’Orbigny in the feeding experiments used prior to the TEM studies. Pre- Fornasini) and epifaunal miliolid Quinqueloculina lamarck- vious biological studies have primarily employed cytoplasm iana d’Orbigny have been recorded moving at rates of less colour and bolus formation as criteria. Colour only works than 50 μmmin−1 (2.4 mm hr−1 or less) in or on sediments, well for calcareous foraminifera with translucent tests (Bern- but move twice as fast on smooth surfaces as in Petri dishes hard, 2000) and often cannot be seen in agglutinated salt (Kitazato, 1988; Travis & Bowser, 1991). This extrapolates marsh species. Bolus formation is not always reliable; an to less than 58 mm d−1 for sediment but up to 116 mm d−1 apertural bolus can persist long after death (Arnold, 1974) on smooth surfaces. The continental slope taxa Cibicides or can represent a post-mortem release of sticky cellular ma- (Cibicidoides) pachyderma (Rzehak) (discoidal) and Am- terial, not a feeding structure (Langer & Gehring, 1993). modiscus anguillae Höglund (spiral) moved at rates of 1–23 We have established several reliable criteria for distin- mm d−1 in culture dishes containing sediment (Bornmalm guishing living specimens of five key salt marsh indicator et al., 1997); A. anguillae, which foraged in a vertical po- foraminifera species, giving most attention to three agglu- sition from a smaller terminal primary aperture, moved tinated species not previously studied. The applicability of moreslowly(maximum20mmd−1). A form and function individual criteria varies among the five species studied: relationship between activity and test shape and size could Trochammina inflata, Entzia macrescens, Miliammina fusca, help explain why some species move more readily and Helenina anderseni,andElphidium williamsoni. Our study more quickly than others. For example, the larger primary shows that the key arenaceous marsh foraminifera are not aperture and higher number of pseudopodia in T. inflata highly mobile, with the largest species, T. inflata moving may explain why it can pull itself over the sediment surface fastest, at ∼2mmhr−1 (50 mm d−1). This maximum move- much faster than expected for its bulky discoidal shape. The ment of the arenaceous species barely overlaps with that re- relationship between motility, aperture location, and pseu- ported for recent studies of estuarine discoidal rotalid forms dopodial production was examined in 22 other species by (e.g., Ammonia tepida and Haynesina germanica)thatmoved Kitazato (1988), who found that having more and/or larger 2–8 mm hr−1, extrapolating to 48–192 mm d−1 (Seuront & pseudopodia gives faster movement than expected in large Bouchet, 2015). discoidal species when compared to streamlined forms like

Siphogenerina (Rectobolivina) raphana (Parker & Jones). dial collections of surrounding detritus, are for food gather- Kitazato concluded that velocity of movement correlated ing, but in some cases, they may have reproductive impor- well with life-form, distinguishing between fast-moving tance or be a normal part of test building. Regardless, de- epifaunal vagile species and slow-moving infaunal vagile tritus around the specimen or at the aperture was the main species from inner neritic waters. For Trochammina inflata, determinant of life status for M. fusca in our study and is movement over smooth culture dish surfaces is probably also applicable for specimens of the other four salt marsh faster than would be expected in loose, fine salt marsh species we examined (see sticky detrital envelopes in Fig. 3). sediments, but it is nevertheless likely to be much faster in Because most individuals of species other than T. inflata natural environments than the other agglutinants studied did not visibly move in the Petri dish, the orientation of here. Overall, our observations suggest that motility (and a specimen was also used to distinguish living cells. Most hence feeding function) is strongly related to test form in individuals orient themselves aperture-side-down on sedi- the epifaunal vagile foraminifera of temperate salt marshes, ment or aperture-side-at-food-source for detached surfaces although other studies show that T. inflata, M. fusca,and such as plant fragments or algal filaments. As a result, spec- E. macrescens can adopt an infaunal microhabitat (e.g., imens were oriented at an angle, and not lying flat in the Goldstein et al., 1995; Milker et al., 2015). dish. This ventral aperture orientation was also noted in Visible motility alone, however, cannot be used reli- unilocular Allogromia sp. Rhumbler by Travis & Bowser ably as the only factor for distinguishing living from dead (1991). In M. fusca, the small, terminal aperture may ne- foraminifera in salt marsh cultures. Temporary dormancy cessitate the ovoidal living specimens often being oriented can also terminate movement (Arnold, 1974), so the other vertically “downward” and its cryptic movements by a rela- non-terminal methods have to be used to distinguish im- tively small number of pseudopodia. Kitazako (1988) also mobile living specimens. Another practical criterion for two found that ovoidal rotalids produced fewer pseudopodia species studied, T. inflata and E. macrescens, is the presence and moved more slowly than discoidal taxa. The orienta- of thick, healthy tests containing visible protoplasm within tion of Trochammina inflata and Miliammina fusca in the one or more chambers. Transmitted light (TL) microscopy Petri dishes is in accord with the feeding observations of can be used to confirm this interpretation by examination of the textularid agglutinant Textularia bocki Höglund on sea- the organic lining contents after acid removal of the agglu- grass leaves (Langer & Gehring, 1993). There are also re- tinated test (Figs. 4A, B). Our studies used TL in a post- ports of this orientation for M. fusca and E. macrescens on mortem context in conjunction with palynological studies dead leaves of salt marsh plants (Murray & Alve, 1999). that use 10% hydrochloric acid and 52% hydrofluoric acid to Langer & Gehring (1993) studied positioning of Textu- extract the organic particles. Acid tests using 1% or 5% HCl laria bocki on seagrass and concluded that glycosamino- on living Trochammina failed to remove the arenaceous test, glycan secreted by pseudopodia was used for “bacterial although 1% HCl dissolved the calcareous tests of Elphidium farming”, forming networks to collect detritus and as- and Helenina. However, Bradshaw (1961) noted that the cal- sociated microbiota. Our new experiments show for the careous taxa Ammonia beccari tepida and Spirillina vivipara first time that agglutinated salt marsh species also secrete Ehrenberg can survive test removal by acid and recover, re- adhesive cellular material around the tests, probably for placing their calcareous shells and reproducing within three gathering (and possibly farming) food particles while rela- weeks after treatment. tively immobile in the salt marsh sediment. Bacteria-fed T. The miliolid M. fusca, however, has a test made of inflata (Fig. 7) often had abundant bacteria inside their tests, much larger sediment grains than the other two trocham- though it cannot be confirmed if this represents colonisation minid agglutinated species. With inverted light microscopy by the bacteria or provides evidence of active collection we determined that light only penetrates the tests of small of bacterial flocs by the foraminifera during “farming”; (<100 μm) specimens. Most specimens of M. fusca taken further studies are needed. Furthermore, strong adhesion from the Chezzetcook marsh mesocosm were >200 μm long. to the sediment surface may be an important characteristic Therefore, seeing cytoplasm within the test was not a practi- in intertidal habitats where transport and removal by tidal cal method of determining life status for this taxon. waters would be a daily hazard. For species with only cryptic movement, such as M. fusca, the best living indications are the rapid attachment (<1 hour) Using Rose Bengal to Validate Live Foraminifera to the bottom of the dish or to algal strands and other large detritus particles, and/or the presence of a detrital bolus Rose Bengal staining is known to over-estimate living at the primary aperture. In experimentally-grown cultures foraminifera (Bernhard, 1988; Murray & Bowser, 2000). of M. fusca, a “rough” granular appearance was noted in Bernhard (2000) gives an extensive review of all known fine-grained (<53 μm) sediment (Goldstein & Alve, 2011). terminal and non-terminal methods for determining live Stereomicroscope examination revealed the grains to be de- foraminifera, including the debatable use of rose Bengal, and trital “feeding cysts” similar to those observed in deep-sea later Bernhard et al. (2006) concluded that the most accurate unilocular species and in the estuarine calcareous Ammonia non-terminal methods are those that use fluorogenic probes. beccarii (Goldstein & Corliss, 1994). These easily detachable, Comparison of the living counts using one of the sev- sticky envelopes are a mixture of detritus, foraminiferal cyto- eral living criteria versus rose Bengal stain counts shows plasmic material, and microbiota (Heinz et al., 2005). They varying results among three temperate salt marsh taxa are normally found at the aperture but can also cover the (Table 2). The difference between living counts based on entire organism (Goldstein & Corliss, 1994). According to culture, compared to staining, were not noticeable for the Heinz et al. (2005), these “cysts”, made through pseudopo- calcareous species Helenina andersoni (3% difference). We

counted more living numbers for the agglutinants T. inflata phagocytosis, the pseudopods can shear off smaller pieces, (c. 8%) and M. fusca (c. 34%) than were stained by rose Ben- which travel into the shell via large vacuoles (Anderson & gal. The large discrepancy for M. fusca, however, possibly Lee, 1991). This could mean that our agglutinated species includes errors in determining living specimens for cultures may also feed on larger phytodetrital particles, breaking of these large, thick-walled, slow moving, detritus-covered them apart before they enter the foraminiferal test. foraminifera where cytoplasm content could not be gauged. Several previous studies concluded that bacteria are the Although M. fusca has a thicker test, rose Bengal absorbs main food for benthic species (Bernhard & Bowser, 1992; easily into cytoplasm not just inside the chambers, but over Langer & Gehring, 1993; Goldstein & Corliss, 1994; Moj- the outside of the test, making the entire specimen bright tahid et al., 2011), though algal food has most commonly pink. This may be because of the “fuzzy” organic linings been fed to foraminiferal cultures for decades (e.g., Myers, seen in Figure 4 (bottom panel), which reflects cytoplasm 1943; Arnold, 1974; Muller, 1975, and reviewed in Lee extruding from micropores over the entire test (see also the & Anderson, 1991). In culture studies of three species of meshwork system of the organic lining in Bender & Hem- Allogromia, Ammonia,andSpiroloculina d’Orbigny, less leben, 1988). Extrusion of cytoplasm may also help with the than five of 28 different species of algae were consumed specimen-covered detritus “glue” previously discussed and significantly, whereas large numbers of bacterial cells were forms the “feeding cyst” structure. consumed (Muller, 1975). Two calcareous species, Ammonia tepida and Haynesina germanica,haveanorange-brown cytoplasm, probably from ingested bacteria and detritus Results of Feeding Trials and TEM (they can consume >25,000 bacterial cells per hour; Moj- Feeding of two agglutinated and one calcareous salt tahid et al., 2011), but the cytoplasm turns green when they marsh species was assessed for the first time by observations are fed the unicellular green alga Chlorella (Moodley et al., of direct feeding, indirect observations of common feeding 2000). Elphidium williamsoni is often epiphytic on algal modes, and by using TEM to determine the contents of di- strands and is a greenish colour due to ingested chlorophyll- gestive vacuoles in T. inflata after feeding trials. Feeding tri- a (Fig. 3G). For saprophagous and bacterivorous deposit als conducted over 12 weeks showed no difference in terms feeders, bacterial “farming” provides the nutrition needed of growth, death, or reproductive events between cultures to support rapid reproduction and growth (Muller & that were fed filtered detritus, cultured salt marsh bacte- Lee, 1969). This may account for the high numbers and ria, or unaltered mesocosm mud. The detritus itself may or small-scale patchiness of foraminifera due to winnowing may not be consumed, as many particles in the food vac- of phytodetrital pieces seen in salt marsh sediments and uoles could not be explicitly identified. Bacteria, in various aggregation of foraminifera at these sites. stages of degradation, were the most common particles in The collection of detritus around the aperture during vacuoles observed with TEM. Therefore, we conclude that feeding is not exclusive to salt marsh foraminifera but detritus primarily gives a physical medium to help grow and conforms to earlier observations (Goldstein & Corliss, gather bacteria on the surface of the foraminiferan’s sticky 1994) for the deep-sea calcareous rotalid taxa, planktonic adhesive (Langer, 1992) from which particles are taken into Globobulimina pacifica Cushman and benthic Uvigerina the shell chamber cytoplasm via the extended pseudopodial peregrina Cushman, and for the shallow-water benthic Am- nets. According to Bowser et al. (1985), vacuoles in pseu- monia beccarii. However, our observations of aperture-down dopodial/reticular networks outside the shell lack digestive re-orientation in M. fusca combined with an apparent lack enzymes, so digestion of the particles only begins once in- of lateral mobility have not been previously reported and side the terminal chamber. Anderson & Lee (1991), however, are possibly important with regard to its patchy habitat suggest that digestion can begin before material enters the in salt marshes where it is concentrated around decaying shell, which would make identification of ingested particles plant stems and leaves of low and middle marsh zones. The by TEM examination of intra-shell material somewhat de- cryptic motility of the salt marsh foraminifera may reflect pendent on the speed of digestion relative to the speed of the abundance of available phytodetritus and associated transport into the shell. Our TEM images of T. inflata show decomposition by bacteria such as Erythrobacter, Agrobac- that still-partly-intact bacteria are present inside food vac- terium,andRoseobacter (Buchan et al., 2003). Some studies uoles (Figs. 5, 6), indicating that most digestion in this ag- report a seasonal cycle for salt marsh foraminifera (e.g., glutinant species occurs inside the test. Buzas, 1965; Horton & Murray, 2006, 2007; Debenay Other cytological studies of foraminifera have rarely ex- et al., 2006; Schönfeld & Numberger, 2007). However, high amined agglutinated species (Bernhard & Geslin, 2018), ex- year-round detrital food availability avoids dependence on cept for unilocular Allogromia sp. (Bowser et al., 1985). In seasonal algal blooms in a temperate marsh with winter ice the multi-chambered species examined, the terminal cham- cover and diminishes the need for energy to be expended on ber and pseudopodial cytoplasm were often filled with many motility. An additional consideration is that rapid changes food vacuoles (Bowser & Travis, 2002). All other studies in salinity can affect the microtubules in the pseudopodia, have examined calcareous species, such as Ammonia becca- which can decrease their movements and ability to “hunt” rii (Goldstein & Corliss, 1994; see most recent rotalid review for food (Pascal et al., 2008). Therefore, in Chezzetcook by LeKieffre et al., 2018). Diatom frustules and bacteria as- Inlet and mesocosm, short-term tidal fluctuations that sociated with clay particles have been found inside food vac- change sediment-water interface salinity many times per day uoles in terminal chambers. Bacteria are apparently digested may negatively impact the use of pseudopodia for actively rapidly, and thus not seen in other chambers (Goldstein & gathering food. This would cause the foraminifera to be Corliss, 1994). If the food is too large to be engulfed by highly dependent on patchy resources, and rapid growth

Figure 8. Schematic of Chezzetcook and its replicated mesocosm salt marsh showing the relationship between zones and distributions of the key foraminifera used for this study. Bar thickness is scaled to the highest percent abundances of the three agglutinants and grouped calcareous species. MHHW = mean high high water; MLLW = mean low low water.

of feeding “cysts” in situ would be most energy-efficient in oratory study methods have used sediment samples in plex- the sediments. This feeding strategy of cryptic movement iglass trays or “antfarms” (Arnold, 1974; Bornmalm et al., in nutrient-rich microzones appears to be a key factor 1997), wherein buried living foraminifera soon emerged at accounting for the fidelity of the marker species for specific the sediment surface, implying their preferred habitat. How- marsh elevations/tidal zones (e.g., Fig. 8). ever, removal from salt marsh sediments that are a crucial The large population sizes of essentially sessile detritus- part of their natural biological/ecological setting is a prob- feeding organisms suggest that they have crucial roles in the lem highlighted by Murray (2006). To precisely define the salt marsh food web (Frail-Gauthier, 2018). This is because niches for benthic foraminifera, one needs to know the spe- these agglutinated forms follow the high patchiness of phy- cific responses to exact abiotic and biotic conditions, and todetritus from salt marsh plants and bacteria. This helps this requires removal from salt marsh sediments. transfer energy up the food web by providing energy-rich Some studies show that the biochemical signature (δ13O patches for the larger meiofauna and small macrofauna that and δ15N isotope ratios) from the foraminiferal tests may consume foraminifera through deposit feeding in the salt reflect their diet more clearly than abiotic factors of their marsh sediment. environment (Mojtahid et al., 2011). Commonly, the abi- Lopez et al. (1979) have shown that in some calcare- otic drivers of marsh foraminiferal distribution are con- ous species on salt marsh mudflats, including Elphidium sidered to be salinity and shoreline elevation (i.e., submer- williamsoni, kleptoplasty plays an important role in their gence/exposure time). However, as a result of feeding on nutrition, and the photosynthetic activity from acquired microphytobenthos, phytodetritus, or bacteria, salt marsh chloroplasts can account for 40–100% of the respiration. foraminifera have different isotopic signatures that reflect The chloroplasts are obtained from microalgae after imme- their different food sources from the mudflat zones through diately digesting the other cellular components (Goldstein to the high marsh (Frail-Gauthier, 2018). In a San Fran- & Alve, 2011). The color of Helenina anderseni in our study cisco Bay estuary, benthic foraminifera responded to bloom suggests that it employs a similar strategy. Chloroplast re- patches of particulate organic matter (POM) much faster tention has the advantage of supplying energy from photo- than other meiofauna, and the distributions and population synthesis during conditions of adequate light, but requires assemblages of foraminifera were probably more related to thin tests, which agglutinant foraminifera cannot produce by food inputs than other environmental parameters (Lesen, their nature. Kleptoplasty may also explain why the calcare- 2005). The fatty acid biochemistry of foraminiferal tests ous salt marsh species are often found epiphytically on al- may also reflect different diets and roles within the ecosys- gal mats at the marsh surface, and only in smaller numbers tem more than environmental or taxonomic differences in the sediment. Calcareous species, such as Ammonia tep- (Suhr et al., 2003). ida, are known to prefer microphytobenthos (e.g., diatoms) In carbon stable isotope studies of the Chezzetcook when at the surface or epiphytic on filamentous algae, but fauna and flora (Frail-Gauthier, 2018), POM had iso- will switch their diet to bacteria if found within shallow sed- topic signatures of −19 (mudflat) to −21‰ (high marsh) iments (Pascal et al., 2008). δ13C, algae had values from −13 to −15‰ δ13C (mud- All observations in this study, however, were constrained flat and low marsh), Spartina had values of −12 to 13 by the artificiality of the in vitro light conditions and the con- −13‰ δ C, and terrestrial C3 plants (middle and high stant temperature of the experiments in microcosm settings. marsh) had values between −25 and −27‰ δ13C. Val- In order to examine individual, living foraminifera in situ, ues for foraminifera ranged between −15 and −25‰ Arnold (1974) designed a field microscope, but this has lim- δ13C; the most depleted values were in the middle and ited practical application for a tidal salt marsh. Other lab- high marsh zones with less Spartina, reflecting their

supposed diets in different zones of the marsh (Fig. 8). Salt Marsh Paleo-Sea Level Implications Calcareous species, consuming cyanobacteria and microal- In addition to the paleoenvironmental implications of the gae in the mudflat had the least depleted isotopic signatures variable δ13C values in salt marsh foraminifera, our high- (<-15‰ δ13C). Within-species variation in detrital and bacte- resolution biological studies of the marsh agglutinants pro- rial feeders, such as M. fusca, also reflected biochemical dif- vide new insight into their use as accurate (±9cmverti- ferences between the lower zones (-16.5‰ δ13C) and higher cal range) markers of sea level datums (Scott & Medioli, zones (-21‰ δ13C) of the marsh. Altogether, signatures from 1980) within a dynamic shoreline environment with diur- sediment POM, plants, algae, and foraminifera reflect the nal tides. The near-sedentary nature and congregating be- transition from marine (less depleted values) to terrestrial haviour of these organisms along with their adhesive cy- sources (more depleted values) which has implications for toplasm are all adaptive features that would tend to allow interpreting paleo-sea levels and salinities in areas where salt these RSL marker organisms to remain within a very narrow marsh cores are not ground-truthed to modern analogs. vertical marsh range (Fig. 8) where they can exploit an am- Finally, food preference in these abundant agglutinants ple food supply below a dense vegetation cover, in contrast has significance for the remineralising of nutrients in the to the dependence of light-requiring organisms (largely the marsh sediment, because foraminifera have a wide variety of calcareous species) on feeding in the open mudflats. feeding modes. Past studies have not determined if different The creation of adhesive feeding “cysts” by abundant salt habits are species-specific or simply characterise functional- marsh agglutinants probably plays a crucial role in binding groups, such as salt marsh agglutinants. salt marsh sediments, in addition to the well-known role of cyanobacterial and algal biofilms (e.g., Amos et al., 1998). Abundance, Biomass, and Foraminiferal Interaction Adhesion and restricted mobility will constrain these agglu- with Meiofauna tinants spatially in the absence of major post-mortem dis- turbance by storms, bioturbation, or freeze-thaw processes, The consumption of bacteria and phytodetritus by keeping the sea-level proxies in place until new sediment foraminifera and interactions with other meiofauna in the buries them and permanently records the biology and ecol- salt marsh sediments is probably important in determin- ogy into the geological record. ing foraminiferal distribution, both on a small-scale and throughout the zones. At Chezzetcook Inlet, plant detritus is much more abundant in the middle and high marsh zones CONCLUSIONS than the low marsh and mudflat zones. Hence in the up- per marsh zones, phytodetritus- and bacterivorous-feeding In vitro cultures and TEM studies of key agglutinated Trochammina and Entzia comprise >75% of abundance and foraminifera and mudflat calcareous foraminifera from the biomass and apparently outcompete other meiofaunal or- cool-temperate region Chezzetcook salt marsh provide new ganisms. The limited motility of M. fusca may help confine insights into living assemblages, forming a basis for refined this species’ dominance to the low marsh. Below this zone, interpretation of the fossil record and paleoenvironments. In calcareous species begin to dominate the foraminiferal as- this study, we have monitored feeding and life-activities of semblages but in lower numbers compared to other meio- three common agglutinated salt marsh foraminiferal species fauna (∼50% abundance; Table 3). using qualitative culturing observations of Miliammina Although we never witnessed predation on meiofauna, fusca and Entzia macrescens, both of which are detriti- some species of foraminifera are thought to be carnivorous, vores/bacterivores displaying only cryptic mobility that con- which could have direct impacts on the meiofaunal ecology tributes to their value as markers of low and high salt marsh of the salt marsh. For example, Ammonia tepida (normally a zones, respectively. Trochammina inflata is a saprophagous deposit feeder on bacteria and algae) is known to also con- bacterivore that has relatively high mobility and a more ex- sume nematodes, copepods, and gastropod larvae (Dupuy tensive marsh distribution (abundant in middle marsh and et al., 2010). Suhr et al. (2008) also noted extensive con- the borders of two other tidal zones). Detritus-gathering at sumption of meiofauna by the large foraminifer Astrammina the aperture is the key method for validating live specimens rara Rhumbler. Although our feeding studies showed no di- in a culture setting. Although no reproduction was observed, rect feeding interactions of T. inflata or M. fusca with meio- we have significantly added to the biological information fauna (Table 1), it is possible that foraminiferal pseudopods needed to determine the niches of salt marsh foraminifera can ensnare and consume small meiofauna. Foraminifera used in high-resolution paleoenvironmental studies, partic- cannot move fast enough to actively hunt down motile ularly as markers of paleo-sea level. The small-scale patchi- metazoa, but for sediments where nematodes are almost ness of these species seems to be dictated by food resources equally abundant, Dupuy et al. (2010) considered it highly and restricted mobility more than favourable abiotic condi- likely that foraminifera could ingest meiofaunal material. tions because the species can thrive in environments with a In salt marsh and mudflat sediments, foraminifera are of- wide range of temperature, salinity, and oxygen levels. ten the dominant meiofaunal species in terms of biomass The TEM studies for examination of feeding modes in and abundance (Table 3), and they form small-scale patches Trochammina inflata show that the species is a sapropelic due to their limited mobility. Because of this, foraminifera detritivore and bacterivore that draws food into the outer could possibly locally deplete microbial resources (bacteria chamber for digestion but also has limited mobility to search and phytodetritus), causing competition with other meio- for areas of optimal food resources. This mixed diet of plant fauna (nematodes, copepods) in the same trophic role as debris and bacteria is likely responsible for shifting the stable foraminifera. isotope C:N values towards terrestrial values and away from

the traditional concept of marine or high-salinity markers. Bornmalm, L., Corliss, B. H., and Tedesco, K., 1997, Laboratory In vitro feeding experiments on interactions between salt observations of rates and patterns of movement of continental margin benthic foraminifera: Marine Micropaleontology, v. 29, marsh foraminifera and associated meiofauna show minor p. 175–184. or no interspecies predation. Biomass measurements estab- Bowser, S. S., and Travis, J. L., 2002, Reticulopodia: Structural lish that at Chezzetcook Inlet, phytodetrital/sapropelic bac- and behavioral basis for the suprageneric placement of granu- terivores (e.g. Trochammina, Entzia,andMiliammina)form loreticulosean protists: Journal of Foraminiferal Research, v. 32, dense populations (>75% of abundance and biomass), while p. 440–447. Bowser, S. S., McGee-Russell, S. M., and Rieder, C. L., 1985, Digestion in more open lower salt marsh zones, kleptoplastic calcare- of prey in foraminifera is not anomalous: A correlation of light mi- ous species dominate the foraminiferal assemblages. croscopic, cytochemical, and HVEM technics to study phagotro- phy in two allogromiids: Tissue and Cell, v. 17, p. 823835–833839. Bradshaw, J. S., 1957, Laboratory studies on the rate of growth of ACKNOWLEDGMENTS the foraminifer, “Streblus beccarii (Linné) var. tepida (Cushman)”: Journal of Paleontology, v. 31, p. 1138–1147. We would like to thank the Cushman Foundation for the Bradshaw, J. S., 1961, Laboratory experiments on the ecology of Joseph A. Cushman Award for Student Research which pro- foraminifera: Contributions from the Cushman Foundation for vided funding for JFG to create the laboratory mesocosm Foraminiferal Research, v. 7, p. 87–105. and conduct stable isotope analyses. Thanks to John Batt Bradshaw, J. S., 1968, Environmental parameters and marsh foraminifera: Limnology and Oceanography, v. 13, p. 26–38. and colleagues at the Dalhousie University Aquatron for Buchan, A., Newell, S. Y., Butler, M., Biers, E. J., Hollibaugh, J. T., building and supporting the marsh mesocosm. We thank and Moran, M. A., 2003, Dynamics of bacterial and fungal com- Dr. Aaron Heiss for all the help in TEM preparations, and munities on decaying salt marsh grass: Applied and Environmental Drs. Sam Bowser, Sue Goldstein, and Emmanuelle Geslin Microbiology, v. 69, p. 6676–6687. Buzas, M.A., 1965, The distribution and abundance of foraminifera for advice with TEM images. Special thanks to JFG’s stu- in Long Island Sound. Smithsonian Miscellaneous Collections, v. dents (Laura Achenbach, Shelby Mclean, Amara Irvine, 149/1, 89 p. David Williams, Robin McCollough, and Zoe Ward) for Chandler, G. 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