Insights into oceanographic environmental conditions such as
paleoproductivity, deep-water temperatures, salinity, ice volumes, and
nutrient cycling have all been obtained from geochemical analyses of
biomineralized carbonate of marine organisms. However, we cannot fully
understand geochemical proxy incorporation and the fidelity of such in
species until we better understand fundamental aspects of their ecology such
as where and when these (micro)organisms calcify. Here, we present an
innovative method using osmotic pumps and the fluorescent marker calcein to
help identify where and when calcareous meiofauna calcify in situ. Method
development initially involved juvenile quahogs (
Biomineralized carbonate of marine organisms such as foraminifera, coccolithophores, and ostracods has provided an abundance of geochemical data critical to our understanding of modern-day oceanographic conditions and processes as well as critical to reconstructions of paleoceanographic conditions and processes. While geochemical proxies of planktic and benthic foraminiferal tests (shells) have yielded copious insights to past sea-surface temperatures, salinity, ice volumes, deep-water temperatures, oceanic circulation patterns, nutrient cycling and paleoproductivity (Katz et al., 2010; Allen and Hönisch, 2012), in the vast majority of cases, initial proxy calibration was developed from core-top sampling and field calibrations. Culturing studies have also contributed greatly to our understanding of the mechanisms controlling these geochemical processes during biomineralization. While we have gained much knowledge on these topics (reviewed by Katz et al., 2010), there remain some significant issues regarding fundamental and emerging proxies. In brief, a variety of factors complicate proxy interpretations; the most common ones in this context include “microhabitat preferences”, “vital effects”, and rapid changes in carbonate chemistry occurring in the uppermost sediment column. Microhabitats refer to the micron- or millimeter-scale distribution of foraminifera with respect to the sediment–water interface, some other physical structure (e.g., worm tube), or chemocline. Vital effects, which can include ontogenetic differences (Filipsson et al., 2010; McCorkle et al., 2008), are physiological processes that impact test geochemistry, although some researchers include environmental processes in the definition of “vital effects” (de Nooijer et al., 2014).
In particular, changes in environmental parameters occurring in the uppermost
part of the sediment might affect proxy reconstruction and it is crucial to
obtain an increased understanding of where in the sediment biomineralization
occurs. For example, one of the most often used temperature proxies,
foraminiferal Mg
Although we know much about where many benthic foraminifera live in sediments
(e.g., infaunal vs. epifaunal; Jorissen et al., 1995; Corliss, 1985) on the
centimeter scale, the true depth habitats and calcification microhabitats of
benthic foraminifera that are used in paleoceanographic reconstructions are
not known. Indeed, as discussed by McCorkle et al. (1990), abundance peaks of
rose-bengal-stained foraminifera are typically several centimeters thick yet
these authors showed species'
To resolve some of these unknowns, we developed a method that will assist in documenting the timing and location of calcification in sediments for calcareous benthic meiofauna. The method employs commercially available osmotic pumps to deliver calcein, which is a fluorescent compound that binds to calcium in biomineralized structures as it is precipitated (e.g., Medeiros-Bergen and Ebert, 1995; Monaghan, 1993; Moran, 2000; Collin and Voltzow, 1998; Hernaman et al., 2000). Using full immersion incubations, calcein has been used to mark bivalves (e.g., Kaehler and McQuaid, 1999; Moran and Marko, 2005; van der Geest et al., 2011) and in laboratory studies regarding foraminiferal calcification (Bernhard et al., 2004; Denoyelle et al., 2012; Dissard et al., 2009; Filipsson et al., 2010; Kurtarkar et al., 2015; Nardelli et al., 2014). In this contribution, we describe a novel point-source calcein dispensation method and show proof of concept for quahog (hard clam) bivalves and benthic foraminifera.
The means used to dispense the calcein are
ALZET® osmotic pumps (Fig. 1a; DURECT
Corporation, Cupertino, CA, USA). Osmotic pumps are devices designed to
deliver pharmaceuticals to animals; as originally intended, they are
installed under the skin of an animal. Different osmotic pump models allow
for different delivery rates and durations. We used model 2ML2 or 2ML4, each with
a reservoir of 2 mL. The 2ML2 was designed to dispense (in mammals) at a
rate of 5 L h
The osmotic pumps were filled with a concentrated solution of calcein
(100 mg L
Visual inspection of an osmopump does not allow for confident assessment of contents. To check if an osmopump continues to dispense calcein, it can be placed overnight, for example, in a clean beaker of seawater. The next day, an aliquot of the seawater can be analyzed with a spectrophotometer. We did such tests early in our investigations to establish accuracy of our calculated estimated dispensation times; results indicated our calculations were adequate (i.e., at our temperature and salinity, the pumps lasted as expected).
Our initial incubations employed juvenile bivalves (quahogs and surf clams;
initially
These containers were initially maintained at 7
Throughout the incubations, living algal food (
Every 2–3 weeks whole specimens (live) were removed from containers noting
their location with respect to the osmotic pump. Specimens were typically
burrowed into the top centimeter. Each bivalve was examined with epifluorescence
microscopy (see below) to determine if they had incorporated the fluorescent
marker calcein. After examination, each individual was placed back in the
sediment near its original location. After
Sediment cores containing benthic foraminifera were collected in May 2013 on
a 3-day RV
As with the bivalve incubations, one calcein-filled osmotic pump was placed
into each core so that the dispensation port was located in the core center.
In most cases, the port was placed just below the sediment–water interface.
In other cases, the port was placed deep (4 cm) below the sediment–water
interface. The cores were maintained at 7
After
All subsequent subsampling of 1 cm intervals was configured in concentric
rings (Fig. 1b, c). Thus, the next 1 cm interval was extruded into the large
diameter ring, three thin-walled plastic rings were concentrically placed
into the core
Epifluorescence microscopy (480 nm excitation; long pass 518 nm emission)
was used to assess calcein incorporation. Preserved materials were examined
with a Leica FLIII stereomicroscope equipped with epifluorescence
capabilities and an Olympus DMP70 digital camera. Whole quahogs and whole
foraminifera, obtained from the
Once imaged at low magnification, select quahog shells were cut with an Isomet slow-speed rock saw (0.4 mm thick blade) to obtain valve cross sections. These valve cross sections had to be polished with fine grit wet/dry sandpaper to obtain a smooth surface. To remove organics, shells were exposed to 3 % sodium hypochlorite for 20 min. After rinsing and drying, these were examined and imaged with the epifluorescence-equipped Leica FLIII stereo-dissecting microscope and DMP digital camera and/or with an Olympus Fluoview confocal laser scanning microscope (CLSM).
The surf clams only survived 1–3 weeks in the cores, but the quahogs
remained active and grew, evidenced by the observations that many
(
Paired micrographs of quahogs after calcein osmotic pump
incubations. Reflected
Because the system used during bivalve incubations was recirculating or
lacking flow, it is important to consider the maximum concentration of
calcein possible if all contents of the osmotic pump were dispensed into the
seawater. We calculate that, at most, the recirculating seawater would have
had 0.02 mg L
In the static (but aerated) setup, the maximum concentration of calcein
possible if all contents of the osmotic pump were dispensed into the seawater
was 0.2 mg L
Some of the calcareous benthic foraminifera in the cores exhibited bright fluorescence while others did not (Fig. 3a, b). As established for the calcein labeling method, the non-fluorescent calcareous specimens either did not calcify during the incubation (Bernhard et al., 2004) or were too far from the osmotic pump port to incorporate calcein. Some of the calcareous foraminiferal tests fully fluoresced (Fig. 3a–d), while others had only one or two brightly fluorescent chambers (Fig. 3e, f). It is possible that the fully fluorescent specimens were the result of reproduction during the experiment (Filipsson et al., 2010; Hintz et al., 2004). In contrast to brightly fluorescent rotalids, entire tests of miliolid (porcelaneous) calcareous foraminifera fluoresced dimly; no agglutinated foraminifera fluoresced (not shown). It is known that at least some miliolids fluoresce after incubation in calcein, even without calcification (Bernhard et al., 2004). It is also established that when rotalid (hyaline) calcareous foraminifera add new chambers, a thin veneer of calcite is precipitated over existing chambers (Erez, 2003; Nehrke et al., 2013). Such a growth habit explains the differential fluorescence patterns in some foraminiferal specimens, where 1–2 chambers are brightly fluorescent and the remainder of the test has less intense fluorescence (Fig. 3f).
Paired micrographs of foraminifera after calcein osmotic pump
incubations. Reflected
As noted for the non-recirculating bivalve incubations, we do not believe the
calcein concentration of the overlying seawater would exceed the minimum
labeling threshold in the foraminiferal incubations even if the entire
osmotic pump contents were released. For foraminiferal calcite labeling, a
calcein concentration of 10 mg L
Unfortunately, the calcein-labeled foraminiferal densities were insufficient to determine the vertical and horizontal extent of calcein diffusion into our muddy sediments. Specifically, calcein-labeled foraminifera were absent from all small-volume radial subsamples of the 0–1 cm interval of one core. Calcein-labeled foraminifera were found, however, in the remaining bulk 0–1 cm interval of the first multicore. Time and resource limitations prohibited full processing of additional multicores; spot checks in those samples did not yield convincing fluorescent foraminiferal calcite. A recently finished master's thesis project confirms the practical use of the osmotic pumps using foraminiferal-laden fjord sediments (Landgren, 2015).
In the course of our method development, a number of lessons were learned. To assist future users of the method, these topics are discussed. Orienting the osmotic pump port downwards is problematic in fine-grained and/or low water-content sediments because conditions likely impede calcein dispensation or the diffusion is too localized to expose many specimens to calcein. It is expected that sediments with high water content (e.g., sediments with “fluff” layers) would not impede dispensation as much as more compacted or consolidated sediments. Attempts to test the osmotic pump port at 4 cm depth did not result in any fluorescent specimens, but we do not know if that was due to lack of calcification or spatially limited calcein diffusion.
To document specifics regarding infaunal calcification horizons, it will be critical to determine the extent of calcein diffusion into sediments. The radius of calcein dispersion and diffusion will vary with sediment grain size, sorting, water content, compaction, hydrodynamics, and community composition (e.g., presence or absence and activity of bioturbators). Diffusion coefficients can be measured directly in sediments (e.g., Krom and Berner, 1980) or they can be estimated from the sediment's formation resistivity factor, which can be estimated from sediment porosity and other sedimentary characteristics (e.g., Ullman and Aller, 1982). Initial verification tests should be considered prior to initiating a lengthy or complicated experiment.
Calculations based on expected dispensation rate, temperature, and salinity can provide estimated duration of calcein efflux. Osmotic pumps are single use; they will not dispense if refilled. Per manufacturer's instructions, osmotic pumps will not perform well if handled without clean gloves.
The cytoplasm of at least one benthic foraminiferal species autofluoresces using excitation and emission wavelengths similar to those for calcein (Apotheloz-Perret-Gentil et al., 2013). The foraminiferal species known to autofluoresce lacks a carbonate test, so it cannot be confused with our calcein-labeling approach. If there are calcareous foraminifera with similarly autofluorescent cytoplasm, distinguishing between cytoplasmic fluorescence (from viability indicators reliant on similar excitation and emission wavelengths) and carbonate fluorescence is not difficult if one considers the patterns and shapes of the signal (Nardelli et al., 2014).
The calcein–osmotic pump method can be used without modification to assess growth rates and calcification locations of juvenile and meiofaunal metazoans with calcareous hard parts (e.g., gastropods, echinoids, ostracods). These units can be deployed in shallow marine waters near shellfish fisheries and in reef areas with sediment pockets. Determining rates of calcification and locations where individuals grow are important to benthic ecology and ocean acidification studies.
As noted, our method will help to better understand foraminiferal
microhabitats. Such knowledge will help to minimize uncertainty and increase
precision in records of paleoceanographic proxies preserved in foraminiferal
tests. For instance, recently the difference in the
Our osmotic pump method can be further modified to deploy these units in
deep-sea sediments using a remotely operated vehicle (e.g.,
While calcein has been used in growth studies for a variety of organisms, to our knowledge, calcein has not been used as a point source to determine calcification in the environment. Most studies using calcein to determine growth rates immerse entire specimens in the laboratory and then release them into nature for later recapture. Our new calcein–osmotic pump approach can help pinpoint where and when meiofaunal organisms calcify in nature. This information is important because, for example, in situ rates of shell growth are not well known.
We thank the captain and crew of RV