Abstract Laboratory cultures of several species of benthic foraminifera were grown under controlled physical and chemical conditions during months-long experiments carried out at the University of South Carolina in 2001 and 2002. A dozen experimental culture chambers contained a c. 1–3 mm layer of trace-metal free silica substrate, and were continuously flushed with water from a large (1600 L) seawater reservoir with known, constant temperature and composition (δ18O(water), carbonate system chemistry, and trace element concentrations). Each year, in most of the culture chambers, one or more species reproduced, producing hundreds of juveniles which grew into size classes ranging from 100 to 500 microns. Bulimina aculeata was the most successful species in the 2001 cultures, and both B. aculeata and Rosalina vilardeboana were abundant in 2002. We determined the shell C and O isotopic composition of the cultured foraminifera, and compared these isotopic values with the water chemistry of the culture chambers, and also with the shell chemistry of field specimens collected from sites on the North Carolina and South Carolina (USA) continental margin. The cultured foraminifera showed substantial offsets from the δ13C of system water dissolved inorganic carbon (−0.5 to −2.5‰, depending on species) and smaller offsets (0 to −0.5‰) from the predicted δ18O of calcite in equilibrium with the culture system water at the growth temperature. These offsets reflect at least three factors: species-dependent vital effects; ontogenetic variations in shell chemistry; and the aqueous carbonate chemistry ([CO3−] or pH) of the experimental system.

The conventional method to distinguish live from dead benthic foraminifers uses Rose Bengal, a stain that reacts with both live and dead cytoplasm. CellTracker Green CMFDA is a fluorogenic probe causing live cells to fluoresce after proper incubation. To determine the more accurate viability method, we conducted a direct comparison of Rose Bengal staining with CellTracker Green labeling. Eight multicore tops were analyzed from Florida Margin (SE United States; 248–751 m water depths), near Great Bahama Bank (259–766 m), and off the Carolinas (SE United States; 220 and 920 m). On average, less than half the Rose Bengal–stained foraminifera were actually living when collected. Thus, while Rose Bengal can significantly overestimate abundance, combined analyses of CellTracker Green and Rose Bengal can provide insights on population dynamics and effects of episodic events. Initial stable isotope analyses indicate that the CellTracker Green method does not significantly affect these important paleoceanographic proxies.

The effect of oceanic pCO2 and carbonate system chemistry on benthic foraminiferal biomineralization and paleoproxy incorporation is not well understood. Carbonate ion concentration is a significant control of stable isotope incorporation in planktonic foraminifera. Moreover, low calcite saturation state, directly related to carbonate ion concentration, appears to alter trace metal proxy incorporation into foraminiferal calcite. Past laboratory benthic foraminiferal culture studies relied on ambient laboratory or compressed medicalgrade air to maintain constant culture seawater pCO2 and ä CDIC with limited success. Improvements made to the system over 15 years (e.g., artificial instead of natural sediment substrate; high volume aeration with atmospheric air; and high volume/low velocity seawater flow) have greatly improved the long-term stability of carbonate system and seawater äCDIC. However, even under stable conditions, we have observed several significant differences in cultured and core-top benthic foraminifera calcite proxy signatures from these two calcification environments (e.g., ä13C, DBa). Current culture efforts are focusing on alkalinity manipulation to experimentally control carbonate ion concentrations and induce biological responses in the recorded stable isotope and trace metal signatures. In as few as 4 months, foraminiferal reproductive events have been observed in supersaturated (Ù ~ 3) and saturated (Ù ~ 1) treatments with greater than 50% of culture populations thriving in the highlyconstrained artificial culture system. Thus, the experimental technique to test carbonate concentration influence on benthic foraminifera stable isotope and trace metal proxies was initially successful. Still, the reliance on atmospheric air for this culture system, with seasonally varying pCO2 and ä CCO2 may cause significant temporal variation in cultured foraminifera paleoproxy Anuár io do Inst i tu to de Geociências UFRJ ISSN 0101-9759 Vol. 29 1 / 2006 p. 459-460

Ontogenetic (developmental stage) measurements of Mg/Ca and Sr/Ca were made on the benthic foraminifer Bulimina aculeata, which were cultured under controlled physicochemical conditions of temperature, pH, alkalinity, salinity, and trace- and minor-element concentrations. We utilized two methods of ontogenetic sampling—whole specimens progressively increasing in length and laser microdissection of a single specimen with subsequent analysis of dissected portions. A novel high-resolution laser-microdissection (HRLM) method allowed for precise (10μm) cuts of the foraminiferal tests (shells) along the geometrically complex sutures distinguishing individual chambers. This new microdissection method limited sample loss and cross-contamination between foraminiferal chambers. Little or no variation in DSr was observed at different foraminiferal developmental stages. Conversely, DMg was enriched during a mid-developmental stage of whole-specimen samples (150–225μm DMg=1.6×10−3) compared to earlier and later stages (<150μm, >225μm DMg=8.3×10−4). Further analysis of HRLM ontogenetic samples showed a larger, age-dependent DMg signature variation. This increase in shell Mg/Ca may contribute substantially to the measured inter-individual variability in Mg/Ca temperature prediction for cultured B. aculeata. Due to relatively large Mg/Ca inter- and intra-individual variability, measuring similar-size foraminiferal samples may improve the precision of paleotemperature prediction. Additionally, partial dissolution of the highest ontogenetically Mg-enriched calcite (DMg=1.3×10−2–1.6×10−2) may occur in undersaturated bottom-water environments or during reductive cleaning procedures. Thus, the calcite phases remaining after partial dissolution by either natural or laboratory cleaning processes may not accurately represent the calcification environment.

Trace/minor element signatures (DCd, DBa, DMg, and DSr) were measured in the tests (shells) of benthic foraminifera cultured in a trace-metal-concentration-controlled system. The culture system was constructed of inert materials and designed to limit microhabitat effects. This system ensured that variation observed in cultured foraminiferal element:calcium (TE/Ca) signatures was due to biologically mediated (vital) effects only. Two species, Bulimina aculeata and Rosalina vilardeboana, reproduced prolifically during two 4-to-8-month culture periods. In every case (i.e., for both species and each element), the inter-individual variability was larger than the analytical precision. Mean (±1 standard deviation) DE signatures for B. aculeata were: DCd: 1.5±0.4, DBa×10: 2.1±0.7, DMg×1000: 0.62±0.15, and DSr×10: 1.5±0.1. Cultured B. aculeata DMg, calibrated from culture and core-top (live) field specimens, predicted temperatures within ±2.0°C. The observed inter-individual variability from culture specimens was as large or larger than comparable results from core-top investigations. R. vilardeboana DCd signatures were significantly lower, while DBa, DMg, and DSr signatures were significantly higher than B. aculeata values. Since our culture system minimizes microhabitat variability, the variation in measured TE/Ca ratios suggests that biological processes are a significant factor in inter-individual and inter-species variability. Comparison of cultured and field-collected foraminiferal DBa signatures supports previous findings that pore-water chemistry is a major environmental influence on foraminiferal test chemistry.

Infauna, including foraminifera and metazoans, were enumerated and identified from five types of seep habitats and two adjacent non-seep habitats. Collections were made with the deep submergence research vessel 'Alvin' from three areas of active seepage in the Gulf of Mexico (Alaminos Canyon [2220 m], Atwater Canyon [1930 m], and Green Canyon lease block 272 [700 m]) and on the Blake Ridge Diapir [2250 m], which is located off the southeastern coast of the United States. The seep habitats sampled included four types of microbial mats (Beggiatoa, Thioploca, thin and thick Arcobacter) and the periphery of a large mussel bed. Sediments under large rhizopod protists, xenophyophores, were sampled adjacent to the mussel bed periphery. A non-seep site, which was >1 km away from active seeps, was also sampled for comparison. Densities of most taxa were higher in the Gulf of Mexico seeps than in Blake Ridge samples, largely because densities in the thick microbial mats of Blake Ridge were significantly lower. Diversity was higher in the Thioploca mats compared to other microbial-mat types. Within an ocean basin (i.e., Atlantic, Gulf of Mexico) we did not observe significant differences in meiofaunal or macrofaunal composition in Beggiatoa versus Thioploca mats or thin versus thick Arcobacter mats. Foraminifera represented up to 16% of the seep community, a proportion that is comparable to their contribution at adjacent non-seep communities. In general, the observed densities and taxonomic composition of seep sites at the genus level was consistent with previous observations from seeps (e.g., the foraminifers Bolivina and Fursenkoina, the dorvilleid polychaete Ophryotrocha).

We describe a novel application of the fluorescent compound calcein (Bis[N,N-bis(carboxymethyl)aminomethyl]fluorescein), which was used to fluorescently label foraminiferal calcite. Foraminifers that were incubated in a 10 mg L-1 solution of calcein and seawater precipitated normal-looking chambers during and after calcein incubation, which lasted up to three weeks. The survival rate, of specimens incubated in calcein was similar to that of control specimens; some specimens reproduced during or after calcein exposure. Thus, this calcein-tagging method is non-lethal. Chambers precipitated during calcein incubation fluoresced a yellow-green when viewed with epifluorescence or laser scanning confocal microscopy (470 nm excitation, 500 nm emission). When viewed alternatively with reflected light, chambers formed after calcein incubation were easily distinguished from calcein-marked chambers, because calcite precipitated after calcein exposure does not fluoresce. Fluorescence is retained through fixation and air drying, thus the signal can be viewed in archived specimens. The method was executed on specimens from 15 species collected from three habitats with diferent environmental conditions. Results indicate that calcein is incorporated by all 15 species. The method has a number of potential applications, including experiments aimed at identifying benthic foraminifers that are faithful recorders of paleoceanographic proxies, as well as field studies to assess locations and chronology of foraminiferal calcification.

We present an apparatus and procedure for culturing deep‐sea (i.e., bathyal) benthic foraminifera under physicochemically constrained conditions. A 1600‐L recirculating culture system was constructed to contribute negligible trace metal contamination; the system was housed in an environmental room maintained near in situ temperatures but at atmospheric pressure. Peristaltic pumps continuously circulated 3 mL seawater min−1 from the reservoir to 9 to 12 culture chambers. A 2‐mm‐thick layer of high‐purity clay‐sized silica substrate was used to minimize the impact of sedimentary microhabitats. Physicochemical parameters (salinity, alkalinity, pH, temperature) varied <2% throughout two culture experiments. Trace metal concentrations were initially set near open‐ocean values and remained constant (i.e., within our analytical precision) during the experiments. Culture seawater was equilibrated with atmospheric CO2, thus dissolved inorganic carbon δ13C varied ~1‰ over the course of each experiment, reflecting the seasonal atmospheric CO2 isotopic variation. Culture seawater δ18O varied ~0.2‰. Each culture chamber was inoculated with as many as 100 foraminifers of single or multiple species. Pre‐existing foraminiferal calcite was identified by fluorescent labeling prior to specimen introduction into culture. The cultures remained viable for >200 d in each experiment and produced up to 2,800 individuals per culture. The growth and reproduction of benthic foraminifera in a physicochemically constrained culture provide a new method for the experimentation and validation of geochemical proxies.