Introduction
The calcification process of the foraminiferal test is the phase of growth
in which elemental and isotopic compositions of the test is determined
and is also the key to generating their morphological diversity. In recent
years, the foraminiferal test has become widely applied as a
palaeoenvironmental proxy, and its geochemical and isotopic composition has
become one of the major tools in palaeoenvironmental reconstructions, even
though the test morphology and chemical composition depend to a certain
extent on the environment (De Nooijer et al., 2014; Schiebel et al., 2017). To this
end, elucidating the detailed mechanisms of foraminiferal calcification has
been treated with great interest in the field of geosciences. For example,
it has been proven by culturing experiments that the seawater temperature
and the Mg / Ca ratio of foraminifera show a strong linear correlation
(Nürnberg et al., 1996; Toyofuku et al., 2000). Meanwhile, it is also known that
the incorporation ratio of Mg / Ca is variable and species specific (summary in
Toyofuku et al., 2011). The chemical distributions, however, vary among even
individuals of the same species and exhibit zonation, corresponding to the
test wall structure (Kunioka et al., 2006; van Dijk et al., 2017). These variations in
chemical composition, both inter- and intraspecific, are inclusively termed
the “vital effect” (Urey, 1951). In order to reconstruct accurate
palaeoenvironments, it is important to utilize reliable proxies, such as the
chemistry and isotopic composition of foraminifera tests. Therefore, the
biological processes of chamber formation is of great importance and
interest.
Observation of the foraminiferal chamber formation process has been reported from as
early as 1854 using the genus Peneroplis (Schultze, 1854), and many species have been
documented thereafter (e.g. Myers, 1935, 1940, 1943; Jepps, 1942; Sliter,
1970; Berthold, 1976; Spindler and Röttger, 1973). Superfine structure
observation by scanning and transmission electron microscopy (SEM and TEM)
have been reported in order to carry out more detailed documentation of the
cellular process of calcite precipitation during chamber formation in the
benthic species Rosalina floridana (Angell, 1967) as well as the planktonic species Globorotalia truncatulinoides (Hemleben et al.,
1986) and Orbulina universa (Spero, 1988). The common features summarized from these detailed
observations on benthic and planktonic species point to the fact that
cytoplasm and the many types of organic sheet-like structures (i.e. organic
layers like outer organic layer, OOL, and inner organic layer, IOL) play
fundamental roles in calcification, as opposed to simple chemical reactions
between calcium and carbonate ions.
Pseudopodium is one of the key features of foraminiferal biology.
Pseudopodia form a part of the cytoplasm consisting of cytoskeleton
structures, such as microtubules and actin filaments, as well as other
organelles like mitochondria, spherical structures, and vacuoles (Marszalek,
1969, reviewed in Travis and Bowser, 1991). Pseudopodium represents a
multi-functional cellular structure serving various purposes such as
locomotion, feeding, digestion, and chamber formation. Foraminiferal
pseudopodia are usually named granuloreticulopodia (see Travis and Bowser,
1991) to define a granular reticulated pseudopodium responsible for feeding,
digestion and locomotion; in the present paper we will simply use
pseudopodia as it is a more general term. The appearance of pseudopodia
changes during chamber formation and a fan-like array of pseudopodia
develops (Bé et al., 1979). Then, an organic structure that forms the
framework for chamber formation, called “anlage”, is formed (Angell, 1967). In
benthic foraminifers, an algal cyst composed of foreign detritus and other
materials is constructed around this anlage (Angell, 1967). Anlage is largely constructed
by foamy and spherical microstructures (< 1 µm) (Angel, 1967;
Hemleben et al., 1986) and is bulging in shape, which led some authors to call it
the “bulge” in early studies using planktonic foraminifers (e.g. Bé et al.,
1979). This bulging anlage is the three-dimensional structure that becomes the
precursor of the chamber. There are three organic layers in the anlage: the one on the
outer surface has been termed the OOL (Spero, 1988), the one in the middle
was initially named the “primary organic membrane” (POM) (Hemleben et al., 1986)
but later changed to “primary organic sheet” (POS) (Erez, 2003), and the
innermost one is called the IOL (Spero, 1988). Precipitation of calcium
carbonate microcrystals takes place on both sides of the POS, sandwiched
between the outer and inner organic layers. In addition to these three
organic layers, the term anlage is now loosely accepted to include the numerous
pseudopodial cytoplasm that are present around them during calcification.
Since different authors have different views and definitions as to what
anlage means (e.g. Angell, 1979; Bé et al., 1979; Hemleben et al., 1986), hereafter we
refrain from using the term anlage and instead use `organic scaffolding' to refer
to the organic framework that the chamber wall is built upon.
In order to investigate the fundamental functions of the POS, the OOL and
the IOL during chamber formation, Nagai et al. (2018) conducted focused ion-beam (FIB) processing on a foraminifera specimen during calcification, which
allows the thin-sectioning of the site of calcification (SOC) without
decalcification to observe cytoplasm and well-preserved morphology of the
calcifying test (calcium carbonate crystals) together using SEM/TEM. Their
observations clearly show that the organic scaffolding has numerous voids
and empty spaces within the membranous structure of the SOC. The presence of
calcification liquid and exo- and endocytosis are inferred, and the growth of
calcium carbonate could be shown using time series samples. However, they
have not documented the processes that lead to the construction of the POS
and other organic structures during chamber formation.
Undoubtedly, the organic scaffolding built prior to chamber formation is an
important factor shaping the characteristic morphology of foraminifera,
serving as a template for calcification. When the foraminiferal test is
dissolved, the organic structure is revealed and it has the same overall
morphology as the calcareous part (Banner and Williams, 1973). Despite the suggestion that pseudopodial activity plays a key role in this
process, little is known about the mechanism. Spindler and Röttger (1973)
first stated that it is pseudopodia that secrete the organic layer
using optical microscopy, working with Heterostegina depressa. However, due to the low resolution
of optical microscopy, they were unable to see the details of the process
and this provided no solid evidence other than speculation.
Although foraminifera are widely used for palaeoenvironment modelling, a
total understanding of the foraminiferal calcification process is still
lacking, impacting the accuracy of predictions made from foraminifera-based
data. An accurate overview and model of the chamber formation by pseudopodia
and the calcification process in calcareous foraminifera is therefore
urgently needed to better our understanding of paleoceanographic proxies. To
fill this knowledge gap, this study aims to elucidate the role of
pseudopodial activities on the formation process of the foraminiferal chamber
and its organic structures within the calcareous wall using
the benthic foraminifera Ammonia beccarii, which has
been used in a few relevant previous studies (e.g. Toyofuku et al., 2017), as a
model system. We combined differential interference contrast (DIC) microscopy
and scanning electron microscopy, capturing DIC images through a time lapse
to document the pseudopodial activities during chamber growth, and we carried
out SEM observations for specimens fixed at different time slices during
the chamber formation process in order to visualize organic structures at the
sub-micron order.
Results
Time series observation with optical microscopy
We were able to observe the chamber formation process of A. beccarii with DIC 59
times in total. Depending on the size of the chamber, it took about 5–8 h to complete the whole process (Table 1). Prior to the start of chamber
formation, exceptional activities were exhibited by the expanded
pseudopodia. Usually for the purpose of feeding and moving, pseudopodia
randomly branches at irregular intervals to arbitrary directions with
variable lengths. During the chamber formation process, however, the
pseudopodial activity significantly differed. A fan-shaped complex
pseudopodial network was constructed (Fig. 1a), expanding from the
aperture of the last chamber. This pseudopodial network is arranged in a
dense, radiating spray resembling that of a dandelion flowerhead. This
unique morphology allowed us to recognize individuals in the beginning of
chamber formation and start our time-lapse observation (from 0 min). For an
average individual, the events of chamber formation can be sequentially
divided into three steps, outlined as follows in a typical time sequence
(see Section “Video Supplement”).
The initial stage of chamber formation was from 0 to approximately 15 min, although this varied considerably depending on the individual and the size of the
newly forming chamber observed, as well as on all stages (Table 1). The initial
stage is the stage where the organic framework for chamber formation is
built. Following the pseudopodial network construction which takes place
from 0 min, an aggregation of cytoplasm (Fig. 1b) quickly became
visible around the aperture of the last existing calcified chamber (Fig. 1b). As the cytoplasm expands, the pseudopodial network retracts to the
surface of the newly forming chamber. We consider the completion of the
organic framework to be the end of the initial stage.
The middle stage of chamber formation took place approximately at around
15–60 min (Fig. 1c–d). During this stage, the foraminifera prepare
the organic scaffolding for calcium carbonate precipitation, which begins
during this stage. By about 30 min, the cytoplasmic aggregation
concentrates in the same shape of a newly forming chamber, like a hemi-sphere
(Fig. 1c). At this point, fine and short pseudopodia have retracted to a
certain extent but are still seen on the surface of the structure. A bright
band, probably representing calcium carbonate starting to become formed, can
be seen on the surface of this. This proceeds to become the chamber wall.
Finally, the pseudopodia retracts inside the forming chamber wall (Fig. 1d).
The late stage of chamber formation is defined as the stage where material,
inferred to be calcium carbonate, is precipitated extensively to thicken the
chamber wall in the newly forming chamber, and took place between around
60 and 400 min (total time varied among individuals; Table 1). We define
the start of the late stage as when the pseudopodia begins to expand again
to cover the organic scaffolding, and also when the whole organic scaffolding is
covered by a layer of calcium carbonate (with pores becoming visible under
light microscopy). At the start of this stage, the pseudopodia expand again
to form a dense network, this time in thicker strands (Fig. 2a). The
length of all pseudopodia appear to be remarkably regular. Calcium carbonate
continues to be precipitated in the forming wall. At this point, the overall
outline of the newly forming chamber is basically fixed. Pseudopodial
movement can be seen inside the forming chamber (Fig. 2a). Cytoplasm
aggregate that filled the newly forming chamber retreats to the previously
formed chamber, leaving an empty space in the new chamber. After that, a
network of pseudopodia is present in the forming chamber, the chamber wall
of which thickens (overall distance between OOL and POS increased over time)
and the pores become increasingly and clearly visible (from Fig. 2b–c;
also see video in Section “Video supplement”). Chamber thickening continues to occur from
this point onwards, generally at around 120–400 min (Fig. 2c). During
this process, the density of the pseudopodial network on the chamber wall
surface increased and wraps the chamber wall like a mesh. As the chamber
wall thickening completes (at approximately 400 min, this is actually quite
variable; the specimen shown in Figs. 1–2 had completed this by 248 min), the mesh-like pseudopodial network on the surface disappears
(Fig. 2d). We consider this to indicate the termination of chamber
formation process. After this, the individual starts to show the usual type
of pseudopodia movement (typical of reticulopodia).
Time series observation of chamber formation by optical microscopy
(continued), as seen in the individual observed on 7 December 2017
(see Table 1). The late stage of chamber formation, where calcium carbonate
is extensively precipitated and the chamber wall is thickened, taking place from
around 60 min after the start of chamber formation (total time varies
considerably among individuals). (a) 65 min, pseudopodia expands again to
form a dense network but in thicker strands than seen in previous stages. (b) 100 min, a network of pseudopodia is seen in the new chamber, the
chamber wall of which thickens. (c) 124 min, chamber wall thickening
continues. (d) 180 min, chamber wall thickening is nearly completed and the
pseudopodial network begins to disappear, indicating that the end of the
chamber formation process is near (actual completion was at 248 min for
this individual). Left: optical microscopy image. Right: the same image with
schematic overlay. Colour legend: deep purple = pseudopodia; light purple = cytoplasm;
magenta = calcium carbonate in the newly forming chamber;
yellow = previously formed chambers.
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Ultra-micro observations on the forming chamber wall
The process of chamber formation is classified into three stages, as
outlined above. Specimens exemplary of each stage were observed with a
scanning electron microscope. Figures 3–6 show microstructures at different
stages of chamber formation seen by the SEM.
Initial stage
In the initial stage of chamber formation, the test was entirely covered
with pseudopodia and organic layer-like structures (Fig. 3a); some parts
of these covering structures were peeled off during the sample preparation
process. Focusing on the chamber being formed, it was possible to observe
the OOL and the POS (Fig. 3b), with the POS being visible from gaps in the
OOL. The interspace between the two layers was narrow (Figs. 3b–c). Even
at high magnification, the outer surface of the OOL itself is a relatively
smooth layer-like structure (OOL in Fig. 3b–c). In some cases, the
pseudopodia can be seen directly expanding from the OOL (Fig. 3c). The
primary organic sheet (POS) can also be observed (Fig. 3b) and is
relatively robust, covered by numerous protrusions. These are convex,
frustoconical structures about 1 µm in width (Fig. 3a–b) and
represent pore plates which corresponds to pores. These were simultaneously
formed when the POS was constructed (green coloured in Fig. 3c–d).
Projections (< µm) were observed on the cytoplasmic surface
of OOL (light green in Fig. 3b). Spherical structures can be seen on the
OOL (blue coloured in Fig. 3C; some appeared crushed, probably due to the
critical point drying process), and similar structures could also be found
on the POS (blue coloured in Fig. 3d and e). The size of these spherical
structures varied from 50 to 500 nm. On the OOL, some elongated
structures, inferred to be pseudopodia, appeared to have a form like that of
a sausage chain (Fig. 3b); the diameter and interval of contractions were
variable. The bulging part contained only cavities, and this form might be
associated with peristalsis. It is known that pseudopodia transport mitochondria
and vesicles (Travis and Bowser, 1991; Cedhagen and Frimanson, 2002), and it
is possible that this peristaltic structure has important roles in such
transportation.
Microstructures during the initial stage of the chamber formation,
shown by SEM images on the left, supplemented by schematic explanation on
the right. (a) Overview of a specimen showing the OOL covering both the newly
forming and older chambers. (b–c) Magnified images showing the OOL and the
POS. (d–e) Magnified image of the POS construction front showing the weaving
action of pseudopodia. (e) The same POS construction front showing the
membranous pseudopodia extending so as to close a large hole (white arrow).
Colour legend: brown = OOL; orange = POS; purple = pseudopodia/cytoplasm; light green = pore
funnel on the OOL; green = pore plate; blue = spherical structures; gray = gap. Thick lines indicate
membranous pseudopodia and dotted lines indicate framework pseudopodia.
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No crystals were found between the POS and the OOL, indicating that no
calcium carbonate has been deposited at this stage, supported by the fact
that the SEM-EDS analyses showed an absence of calcium signals (Fig. 6a).
Microstructures during the middle stage of the chamber formation
shown by SEM images on the left, supplemented by schematic explanation on
the right. (a) Overview of the ventral side of a specimen, showing the
cytoplasm covering the newly forming chamber. (b) Magnified image showing the
OOL on the suture, between the new chamber and the previous chamber. (c) A
higher magnification image of the POS showing spherical structures on the
POS. (d) Image showing the matching relationship between convex structures on
the cytoplasmic surface of the OOL and the pore. (e) Needle-like crystals of
calcium carbonate between the IOL and the OOL, with white arrowheads indicating
clear gaps between the crystals. Colour legend: brown = OOL; orange = POS; purple = pseudopodia/cytoplasm; light green = pore funnel on the
OOL; green = pore plate; magenta = calcium carbonate; blue = spherical
structures; gray = gap. Thick lines indicate membranous pseudopodia and
dotted lines indicate framework pseudopodia.
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Middle stage
At the middle stage, the interspaces among the framework structure
constructed by the pseudopodia has been filled to a much larger extent than
in the initial stage, with much fewer gaps (about 5–200 nm; grey
coloured in Fig. 4b and c) that could be seen. Nevertheless, calcium
carbonate precipitation has already started between organic layers in some
parts of the forming chamber (Fig. 4d). Upon closer observation, these
were revealed to consist of needle-like structures that covered the surface
of the POS, close to the previously formed chamber. These needle-like
structures were confirmed to be crystals of calcium carbonate precipitating
vertically between the OOL and the POS by EDS observation (Fig. 6b).
Therefore, the precipitation does not start at the same time across the
entire chamber, but instead begins locally right after the completion of
organic layer construction. At this point, there are still small gaps
between independent crystals. It can also be noted that the framework
structure formed by pseudopodia appears to have a certain directionality in
growth.
Microstructures during the late stage of the chamber formation
shown by SEM images on the left, supplemented by schematic explanation on
the right. (a) Overview of the dorsal side of a specimen, with the newly
forming chamber on the bottom. (b) Magnified image of the OOL seen from the
outside. (c) Image showing a cross section through the forming chamber wall.
(d) A magnification of the IOL seen from the inner side, showing pores and
lots of spherical structures. Colour legend: brown = OOL/IOL; orange = POS; purple = pseudopodia/cytoplasm; light green = pore funnel on the
OOL; green = pore plate; magenta = calcium carbonate; blue = spherical
structures.
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Numerous, rather regularly spaced pores (about 1 µm) can be clearly
observed on the crystalline layer (Fig. 4a). In the part where OOL was
curled up to reveal the inner side (see Fig. 3b), convex structures
corresponding to pore lining were seen (Fig. 4d). This has been termed
“pore funnel” by Hottinger (2006), a term that we adopt here. Interestingly, pores
cannot be seen at this stage from the outer side on the OOL with SEM
observation (Fig. 4a–b), and the OOL appears to be entirely smooth in the parts
where the framework has been filled completely. We interpret this as being due to
a layer of cytoplasmic material that also fills the pore lining (i.e. the
“well”) during chamber formation, which regresses after the completion of
chamber formation (and therefore becomes visible under SEM). As discussed
previously, however, pores can still be seen during chamber formation using
light microscopy due to the semi-transparent nature of the organic layers as
well as the thin calcium carbonate layer. Algal cysts including Dunaliella individuals
can be seen overlaying the OOL (Fig. 4a). The OOL is a continuous
structure that envelopes the entire test, and it extends to the newly
forming chamber from the aperture of the previously formed chamber. In some
parts where calcium carbonate precipitation has not yet taken place, the
outer surface of the POS can be seen (Fig. 4a and c) and like in the
initial stage, many frustoconical structures about 1 µm in width are
seen (Fig. 4c). Spherical structures (blue coloured in Fig. 4b–c), about
50–500 nm in size, could be seen on both the OOL and the POS as in the
initial stage.
Elemental maps of cross sections through the forming chamber wall
at different stages, shown by SEM-EDS analyses. (a) Initial stage. (b) Middle
stage. (c) Late stage. White lines indicate the position of the POS. The
false colour maps indicate the intensity of calcium signals, corresponding to
the legend shown on the bottom. Thick lines indicate membranous pseudopodia
and dotted lines indicate framework pseudopodia.
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Late stage
In this final stage (Fig. 5), the construction of organic layers has been
fully completed, and a layer of calcium carbonate began precipitation across
the entire newly forming chamber. The OOL is therefore seen as uniformly smooth
and without gaps, from the outer side (Fig. 5b). A cross section through the
forming chamber wall at the late stage clearly shows three completed layers
(corresponding to the IOL, the POS, and the OOL, respectively, from the inner
layer outwards, in that order) and two layers of precipitating calcium carbonate
sandwiched between the IOL and the POS as well as between the POS and the
OOL (Fig. 5c). EDS analyses obtaining signals of Ca, C, and O
simultaneously (Fig. 6c) clearly indicated high Ca signal distribution
being detected these two layers, showing that these layers are calcium
carbonate in nature.
The precipitation of calcium carbonate crystals, continuing from the middle
stage, leads to carbonate crystals becoming increasingly densely packed,
with gaps between crystals completely disappearing by the end of the late
stage (which marks the end of chamber formation). In the figured specimen
observed in Fig. 4, the thickness of the calcium carbonate layer is about
1 µm between the OOL and the POS, and about 0.3 µm between the POS
and the IOL.
As in the middle stage, the exterior of the OOL appears to be smooth (i.e. pores
cannot be seen yet) (Fig. 4d). The IOL, however, when seen from the
cytoplasm side, is seen to be covered by regular depressions that
correspond to the convex side of the pore plate on the POS (which may be
named the “inner pore”) (Fig. 5d). The IOL can therefore be considered to
have the same shape as the POS. Spherical structures could also be observed
in the late stage on the surface of the OOL, but the size of these structures
was more variable than in the earlier stages, ranging from 50 nm to 1 µm (Fig. 4c–d). Furthermore, similar structures could also be observed on
the IOL (not shown).
Schematic illustrations of chamber formation. (a) Construction of
organic layers by pseudopodial weaving and subsequent gap-filling. (b) The
entire chamber formation process from the initial stage on the left side to
the late stage on the right side. Colour legend: brown = OOL/IOL; orange = POS, purple = pseudopodia/cytoplasm; light green = pore funnel on the
OOL; green = pore plate; magenta = calcium carbonate (see Fig. 5e);
blue = spherical structures; gray = gap.
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Discussion
The weaving of organic layers during chamber formation
This study is the first to observe the detailed making of organic layers
during chamber formation and revealed that the layers are actually woven by
pseudopodial activity. A schematic diagram is presented in Fig. 7, which
outlines the general observations. The basis of organic layer formation is
the interweaving of a pseudopodial framework (Fig. 7a), the interspaces of
which is then filled in with a further layer of pseudopodial material,
resulting in a complete organic layer. The pseudopodia are observed to form
a dense framework (purple dotted lines in Figs. 2a, 3 and 7), which is
then overlaid by a layer of membranous pseudopodia which fills the
interspaces (Fig. 3e). In the OOL, numerous spaces of 100 nm–1 µm
can be seen (gray in Figs. 3, 5, and 7), which represent the interspaces
between the framework that is yet to be filled. In some instances, the
membranous pseudopodia were observed during the process of filling the
interspaces, sometimes from more than one direction (e.g. Fig. 3e), by a
gradual, webbed expansion. In short, a framework is constructed by a
pseudopodial network, which is then overlaid and the interspaces filled in
by a layer of membranous pseudopodia. According to a recent study (Nagai et
al., 2018), the three organic sheets (OOL, POS and IOL) initially appear to be
independent even at the very early stage of chamber formation, when the
total thickness of the whole organic sheet being less than 1 µm. We
expect that these organic sheets themselves are ultimately expanded from a single
root, but separate branches of pseudopodia form each organic sheet.
The importance of organic layers in the early stages of chamber formation
has been speculated in previous studies, but little was known about its
origin. It was previously thought that the organic layer was secreted from
the pseudopodia (e.g. Angell, 1967; Röttger, 1974; Hemleben et al., 1986),
and Spindler and Röttger (1973) reported that the organic layer seems
to be connected with pseudopodia. These studies were largely limited in that
their magnification (only light microscopy was available then) was not
sufficient in resolution to observe the detailed process. The process
documented herein provides evidence for an entirely novel model in that the
pseudopodia themselves weave the organic layers (Fig. 3c–e,
Section “Video Supplement”) – in other words the organic layer is part of the cytoplasm.
The reason why the space between IOL and POS is narrower than between OOL
and POS (meaning the inner calcareous layer is thinner than the outer) is
presumably caused by the difference in the growth rate of calcareous material
between the inner side and the outer side. Assuming that the materials for
chamber formation are transported from the seawater, it can be presumed that
the inner side will become thinner because the chamber wall is formed and
material transportation is more restricted on the inner side.
Pore formation
Fine-scale observations from the present study allowed us to reconstruct the
actual steps in pore formation. As shown already in previous studies (Bé
et al., 1979; Spero, 1988), the structure known as a “pore” in foraminifera is
actually a composite structure formed by two opposing wells converging at
the POS, one opening towards the outer side located on the OOL and one
opening towards the cytoplasm side located on the POS (and same on the IOL).
The POS–IOL well has been called the pore plate in previous studies (e.g.
Haynes, 1981). These pore plates can also be seen on the organic layer
template when fossil foraminiferal tests are dissolved (Bannar et al., 1973;
Banner and Williams, 1973; Hottinger and Dreher, 1974; Cadre et al., 2003;
Ní Fhlaithearta et al., 2013). Pore plates seem to be dented on the IOL
side, according to a previous study (see Fig. 3c in Nagai et al., 2018). Therefore,
the pores are not actually pass-through structures formed at once but are
instead formed in unison by separate processes on the OOL and the IOL. Pores
have been suggested to be used for respiration (e.g. Berthold, 1976;
Leutenegger and Hansen, 1979). As O2 and CO2 used in respiration
are nonpolar molecules, they are able to pass through the cell membrane. As
such, the existence of pore plates made from cytoplasm seen in the present
study should not influence respiration. Our observations show that in the
initial stage of chamber formation, the pore plate (visible as frustoconical
structures of about 1 µm) is already present when the POS is woven, at
the growth front. Pore funnels, about 0.5 µm in size, which pair up with
the pore plate in the same location (but open to the opposite direction), are
formed on the OOL. This structure and the pore plate collectively form the
pore, and there is no space between the two for calcium carbonate to
precipitate, and therefore the pore is not calcified. Pore plates and pore
funnels smoothly peeled off from one another (Figs. 3b and 7a), suggesting
that pore plates and pore funnels belong to independent organic sheets
formed from separated pseudopodia. Pore plates and funnels were gently
adhered to each other before calcification started. It is thought that pore
plates and pore funnels are formed simultaneously face to face, during the
organic sheet formation. All hyaline foraminifera that have been observed in detail
possess pores. Since pores are not pass-through and are formed as the framework
for the organic layer (i.e. OOL, POS, and IOL) formation is woven, and since the
layers are somewhat flexible before calcification, one possible speculative
function for pores is to serve as a connective structure between OOL and
IOL. In this scenario, the pores “staple” the organic layers of the forming
chamber together, so that the sites of calcification maintain a consistent
thickness and form throughout the chamber while calcification occurs.
Spherical structures
The existence of spherical structures on the surface of organic layers have
been reported in previous studies (Angell, 1967; Spero, 1988), but their
function and significance have not been mentioned. A recent study (Nagai et al.,
2018) utilized focused ion beam (FIB) technology to process SEM samples
in order to visualize calcium carbonate and organic layers on the same
semi-thin section. They were able to observe the presence of spherical
structures in the site of calcification, and they might be responsible
for exo- and endocytosis. The spherical structures increase the surface area
and probably serve to improve the material exchange efficiency, by
increasing the contact surface area with seawater. In the present study, we
could observe numerous spherical structures on all three organic layers,
including the OOL, the POS, and the IOL. This indicates that the spherical
structures probably play important roles in material exchange during
calcification for both the outer and inner calcified layers, and as the
spherical structures are inferred to result from the activity of the organic
layers this further strengthens the active role of these layers in
calcification (i.e. they are not mere templates). The number of spherical
structures increased as the chamber formation progressed. There is a
variation in the size of spherical structure from ca. 50 nm to 1 µm.
Small ones were relatively more numerous at the initial stage and large ones were relatively more numerous at the late stage, but all sizes of the spherical structures
are found across all stages.
Prospects for calcification model
Until now, the exact process of calcium carbonate precipitation, in terms of
how precipitation was related to the degree of isolation of the site of
calcification, remained largely unclear (Erez, 2003; De Nooijer et al., 2014). In
the present study, the sequence of events during calcification was made
clear by time-series observations, and importantly both the formation of
the organic layer and calcium carbonate precipitation were observed together. It
is significant that during the middle stage, although the overall shape of
the forming chamber has already been formed by framework-like pseudopodia,
the precipitation of calcium carbonate was seen to initially start before
the framework pseudopodia have been fully covered and filled by membranous
pseudopodia. The organic layers (especially well-observed in the OOL and the
POS) still contained numerous gaps < 1 µm in size, which we
interpret as maintaining the exchangeability of seawater and elements contained
within, for the initial part of calcium carbonate precipitation. The site of
calcification is therefore interpreted to be still open to seawater during the middle stage.
In the late stage, however, the organic layers have been completely filled
by membranous pseudopodia and no such gaps remain. At this stage, therefore,
the site of calcification is closed from the surrounding seawater. Hence, we
interpret that during the late stage the elements required for calcification
must be selectively taken up by biological means such as exo-endocytosis or
ion pumps through the OOL. Although we could not observe the IOL in detail during this process
(due to its position below the POS), the IOL most likely
receives the required elements through pseudopodial transport during the
late stage, although whether this originates directly from the forming
chamber or the previous chambers cannot be ascertained yet. Previous
evidence (e.g. Toyofuku et al., 2008; De Nooijer et al., 2009) appears to suggest that
calcium and carbonate are transferred from the cellular material inside the
previously formed chamber. The elemental composition of the inner calcified
layer, formed between the IOL and the POS, is probably more closed and
strongly affected by cellular processes compared to the outer calcified layer
between the OOL and the POS. Therefore, the magnesium contents of the inner
layer may differ from the outer layer and pure calcite may be precipitated
in the inner side. The POS has been widely considered to be the only
template for calcification (Hemleben et al., 1986), but recent research has
revealed that calcium carbonate precipitation also occurs on the other
organic layers (Nagai et al., 2018). It was also shown that the POS gradually
becomes obsolete as the chamber matures towards completion of thickening.
Therefore, the true role played by the POS during calcification should be
reconsidered. A likely function of the POS is that by doubling the surface
area on which precipitation occurs, the existence of the POS doubles the
rate of chamber formation. Considering that the mobility of foraminifera is
highly limited during chamber formation, increasing the efficiency of
chamber formation is probably beneficial and adaptive for the foraminifera.
It is well known that the chemical and isotopic compositions of calcareous
foraminifera tests differ significantly from those precipitated
inorganically, and the compositions also differ among different species.
This effect is collectively known as the “vital effect” (Urey et al., 1951) and
has been a great hindrance to the use of foraminifera tests as geochemical
proxies, for example to reconstruct palaeoclimates. In attempt to explain
the vital effect, Nehrke et al. (2013) proposed a transmembrane transfer–passive transfer (TMT/PT) model by observing Mg / Ca ratio during
calcification, assuming that a low ratio indicates active transport (i.e.
transmembrane transfer sensu Nehrke et al., 2013) and a high ratio indicates passive
transport, as Mg is discriminated against in Ca channels in active transport.
Their observations indicated that passive transportation predominates at the
early period of calcification, with active transport becoming dominant at
later periods. This is consistent with the results outlined above from our
observations during the present study, but we were able to reveal the
reasons behind the differences in Mg / Ca ratios in early and later periods of
calcification, which is that during the middle stage the site of
calcification has not yet been fully isolated from the surrounding seawater. This is a key
finding with respect to what actually causes the vital effect, in that the
construction process of the organic layers can significantly influence when
the site of calcification becomes isolated, leading to differences in
chemical and isotopic compositions of the test by the proportion of
contributions from passive vs. active transport. The elemental analysis of
fluid at the site of calcification, however, is still currently unmeasurable
due to technical limitations. Nevertheless, because magnesium ions are an
inhibitor of calcification, it can be speculated that during
biomineralization magnesium ions are actively discriminated and removed from
the fluid at the site of calcification (Zeebe and Sanyal, 2002). Therefore,
it is presumed that calcite with low Mg / Ca precipitates even around the POS.
It is reported in many species that the foraminiferal Mg / Ca is high around
the POS, but it is still much lower than Mg / Ca estimated from inorganic
precipitation experiments (De Nooijer et al., 2014). The elemental
partitioning in foraminiferal tests must be strongly controlled through the
elemental composition of the fluid in the SOC, which is a key subject for
future studies.