Merging imaging technologies and metabarcoding to characterize the Rhizaria

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Silica and the marine silicon cycle

Silicon (Si) is the second most abundant element in Earth’s crust. Silicon atoms bond with oxygen (O) atoms to create silicon dioxide (SiO2), also known as silica, which may be either crystalline or amorphous. This element cycles through the marine environment (Figure 1), entering the ocean primarily from rivers. Other secondary sources include submarine groundwater, erosion of marine soils, hydrothermal vents and aeolian inputs. In the ocean, Si is essentially found as dissolved silicic acid (dSi), which is required for the growth of some phytoplankton groups like diatoms and other silicified planktons, such as radiolarians and phaeodarians, sponges, silicoflagellates and several species of choanoflagellates. These organisms, commonly called “silicifiers”, remove dSi from the ocean to build their cell tests. After they die, their test acts as ballast, thus causing themto sink toward the ocean floor. While sinking, most of the Si recycles throughout the water column, being again available as dSi for other silicifiers. The fraction of the cells that resists dissolution reaches the sea-floor, where they can either remain, forming a siliceous ooze, or dissolve and return to the upper layers of the ocean through upwelling processes.
The Si cycle is intimately connected to other biogeochemical cycles, like those of carbon and nitrogen. These cycles interact via marine primary production, which drives atmospheric CO2 sequestration to the deep ocean via the biological pump and, ultimately, exerts control over the Earth’s climate (e.g., Buitenhuis et al., 2006; Reynolds, 2001).
Among organisms that require Si to grow, diatoms have been the main focus so far. The production of bSi in the oceans by diatoms has been estimated to 240 Tmol Si yr-1 (Nelson et al., 1995). Diatoms largely dominates all other biogeochemical fluxes in the Si cycle (Tréguer and De La Rocha, 2013). However, the contribution of other silicifiers, like sponges and Rhizaria, is poorly documented.
Since the late 20th century, silicified rhizarians have drawn attention for their role in the marine Si cycle (Heath, 1974). Takahashi (1983) suggested that, in some oceanic areas, the daily flux of rhizarian biogenic silica (bSi) ranges around 20% to 30% of the overall bSi.
Recent studies combining genomic and in situ imaging data have evidenced that densities of siliceous Rhizaria, have so far been underestimated. Biard et al. (2016) reported that some Rhizaria taxa represent approximately 33% of the large zooplankton (>600 µm) in the upper water column. Guidi et al. (2016) pointed out Rhizaria’s significant involvement in the export of C to the deep ocean, with their abundances correlating with export C fluxes at 150-m depth in oligotrophic oceanic regions. These studies have raised awareness of the global significance of Rhizaria in the biological pump, as well as in the Si cycle.

Rhizaria, conspicuous organisms in the ocean

Oceanic Rhizaria are a very diverse microplankton group that have existed at least since the Cambrian era (~500 million years ago). This group of protists is a major lineage of eukaryotes, including Cercozoa and Retaria (Cavalier-Smith, 2002), with the latter grouping Radiolaria and Foraminifera (Figure 2).
Figure 2. Schematic of the eukaryotic tree based on a consensus of phylogenomic together with morphological and cell biological information. SAR is the conglomerate of Stramenopiles, Alveolates, and Rhizaria, which together make an assemblage encompassing perhaps half of all eukaryote diversity. CRuMS is an amalgamation of several ‘orphan’ taxa: the Collodictyonids, Rigifilida, and Mantamonas. Adapted from Keeling and Burki (2019).
Essentially unicellular though some are capable of forming colonies up to over 1 m in length, Retaria span a wide range of sizes, from tens to hundreds of micrometers (Boltovskoy et al., 2017).
Some species of Retaria have elaborate mineral skeletons of strontium sulfate (Acantharia), calcium carbonate (Foraminifera) and opaline silica (Nassellaria, Spumellaria and Phaeodaria). Radiolaria are divided into two major lineages: the Polycystinea (including the three orders: Nassellaria, Spumellaria and Collodaria) and the Spasmaria (including Acantharia and Taxopodida; Krabberød et al., 2011). Phaeodaria, initially classified in Radiolaria, is now placed among the Cercozoa as revealed by molecular phylogeny (Polet, 2004).
The work performed during this thesis focuses on the silicifying Rhizaria (polycystine Radiolaria and Phaeodaria). The study of these planktonic protists has been essentially used for paleoceanographic reconstructions, based on the fossil record left by their silicified skeletons in oceanic sediments (Matsuzaki et al., 2014; Moore, 1978). Almost all polycystine species preserve well in the bottom sediments while most Phaeodaria skeletons dissolve more readily before reaching the sea-floor (Takahashi, 1983).
Despite their potential role as Si consumers and C exporters (e.g., Lampitt et al., 2009; Takahashi, 1983) in the marine environment, very little is known about their physiology, life cycle and ecology, particularly regarding silica. This is essentially due to the difficulty of culturing Rhizaria, as well as the delicate morphologies of living specimens which make them liable to collapse when using conventional sampling methods.

Cell structure

Polycystine radiolarian and Phaeodaria skeletal morphology can be very variable depending on the taxa considered (Figure 3). Generally, their ornamented skeletons surround a central and porous capsule from which pseudopodia (long and slender cytoplasmic projections) radiate.
Skeletons in protists are believed to play a role in essential functions. For instance, for the microalgae diatoms it has been proven that the skeleton provide mechanical protection for the cell against predators (Finkel and Kotrc, 2010; Hamm et al., 2003), as well as an effective pH buffer (Milligan, 2002). Less is known about the role of the Rhizaria’s skeletons, but it is likely to improve the uptake or storage of bio-essential elements (Suzuki and Not, 2015).
(a) Nassellarian of the family Plagiacanthidae, (b) Spumellarian of the superfamily Stylodictytoidea, (c) Spumellarian of the superfamily Spongodiscidae, (d) Phaeodarian of the family Challengeridae (Challengeron sp.),
(e) Detail of central capsule of a colonie of Collodarian, family Sphaerozoidea, (f) Phaeodarian of the family Aulacanthidae (Aulacantha scolymanta), (g) Phaeodarian of the family Coelodendridae (Coelechinus sp.). Pictures N. Llopis Monferrer except picture (e), taken by A. Leynaert.
Polycystine Radiolaria. Most Nassellaria, Spumellaria and siliceous Collodaria possess solid and dense skeletons of amorphous silica, so-called opal (SiO2) (Figure 4).
Nassellaria present a heteropolar skeleton with one or more sections aligned along an axis. From the narrower end, the cephalis, which is the first to be formed, several segments succeed: cephalis, thorax and abdomen (Boltovskoy et al., 2017). Skeletons vary from simple tripods to elaborate, helmet-shaped structure, often with spines or other ornamentations (Boltovskoy et al., 1990).
Most Spumellaria have radial or spherical symmetry with centrifugal shell-growth, but their skeleton can also be flat or elliptical (Suzuki and Not, 2015). The nucleus is located in the central capsule and it is surrounded by radially arranged lobes of cytoplasm, enclosed by a porous capsular wall.
Collodaria is the only taxon with colonial representatives, which can be composed of tens to thousand cells. Each colony consists of a spheroidal, hollow, gelatinous envelope containing numerous interconnected cells embedded within a gelatinous matrix (Anderson et al., 1987). Cells can be either naked (e.g., Collozoum sp.), or surrounded by a porous, spherical Si skeleton (e.g., Collosphaera globularis) or provided with siliceous spines embedded in the cytoplasm (e.g., Sphaerozoum sp.).
Phaeodaria. Phaeodarians are often larger than polycystine individuals. Their cell size ranges from several hundreds of micrometres up to several millimetres. Phaeodaria’s skeleton is also composed of amorphous silica but is more porous and less solid than polycystine’s skeletons (Nakamura et al., 2018). The porosity of their skeleton and the organic matter content in their test is presumably responsible for the poor preservation of these organisms in the fossil record (Takahashi et al., 1983). As for Radiolaria, their geometry is complex and varies among families. This group is mainly defined by a central capsule containing the phaeodium, a mass of partially digested food, generally darkly coloured that emanates from the astropyle, which resembles an oral aperture (Figure 4). Phaeodarians possess a double-walled central capsule and the skeletal network can be surrounded by filopods.
Figure 4. Scheme of the skeletal elements of the shell of a typical Nassellaria, Spumellaria and Phaeodaria (Adapted from Boltovskoy et al., 2017). The right-hand scheme refers to the soft parts compared to the left-hand one, which refers to the skeleton alone.

General biology and ecology

Place in the trophic network. Despite the ubiquity of these organisms in the oceans, fundamental information about their feeding behaviour is poor. These protists are mainly heterotrophic, as they can capture preys through adhesion to their pseudopodia.
Polycystine consume a wide variety of prey, from bacteria and algae up to small invertebrates (Gowing and Coale, 1989). Beside the heterotrophic behaviour, many polycystine inhabiting surface waters, exhibit symbiotic microalgae providing nutrients to the host (Decelle et al., 2015). Phaeodarians are omnivorous, they can feed on other plankton or on organic suspended matter in the water column (Gowing, 1986). Unlike many polycystine, no Phaeodaria have been reported harbouring microalgal symbiont so far.
Very little is known about the predatory pressure on Rhizaria, although there are evidences of other plankton such as Foraminifera, salps and small crustaceans preying on Rhizaria (Gowing 1989; Swanberg, 1979).
Reproduction. Rhizaria are difficult to keep alive in cultures and our knowledge about their life cycle is incomplete (Suzuki and Not, 2015).
For several species of Nassellaria, Spumellaria and Collodaria it has been observed that after organisms have turned whitish, they release small bi-flagellated cells generally called swarmers (Suzuki and Not, 2015).
For Collodaria, binary fission within the central capsule has been reported as well as swarmer production (Anderson and Gupta, 1998; Biard et al., 2015).
Phaeodaria species have also been observed to reproduce by cell division and swarmer production (Hughes et al., 1989), but the entire life cycle has never been replicated in the laboratory.
Data about the longevity of Rhizaria is limited. Based on laboratory observations, it seems that Rhizaria can live from several weeks to several months, likely depending on the taxa, before reproducing (Boltovskoy et al., 2017).

Silicification

The general process of silicification has been essentially studied for diatoms. This complex process involves the transport of Si (mediated by Si transporters or by diffusion) across the cell membrane and then through the cytoplasm to the site of polymerisation within the silica deposition vesicle (SDV) (Martin-Jezequel et al., 2000; Thamatrakoln and Hildebrand, 2008). Although the presence of Si transporters that enable the uptake of dSi from the environment have recently been reported in rhizarians (Marron et al., 2016), the physiological and morphological factors regulating Rhizaria skeleton morphogenesis are poorly documented.
One of the first statements about the Rhizaria skeletal secretion was made by Haeckel in the 19th century:
“It may indeed be assumed that these skeletons arise directly by a chemical metamorphosis (silicification, acanthinosis, etc.) of the pseudopodia and protoplasmic network; and this view seems especially justified in the case of the Astroid skeleton of the Acantharia, the Spongoid skeleton of the Spumellaria, the Plectoid skeleton of the Nassellaria, the Cannoid skeleton of the Phaeodaria, and several other types. On closer investigation, however, it appears yet more probable that the skeleton does not arise by direct chemical metamorphosis of the protoplasm, but by secretion from it; for when the dissolved skeletal material (silica, acanthin) passes from the fluid into the solid state, it does not appear as imbedded in the plasma, but as deposited from it However, it must be borne in mind that a hard line of demarcation can scarcely, if at all, be drawn between these two processes.” [Haeckel, 1887, p.CXXXIV] Anderson (1981, 1994) suggested the presence of SDV in polycystine radiolarians, similar to that found in diatoms. Recent studies have used a fluorescence compound, the PDMPO ((2-(4-pyridyl)-5-[(4-(2-dimethylaminoethylaminocarbamoyl) methoxy)-phenyl] oxazole) to elucidate the silicification process in Rhizaria. This compound binds with Si under acidic conditions, emitting a green fluorescence under ultraviolet light, that allows the imaging of newly deposited Si. These studies have also suggested the deposition of Si in an acidic compartment, likely a SDV (Ogane et al., 2010, 2009).
So far, three silicification processes have been reported for Polycystine radiolarians (Anderson et al., 1987; Boltovskoy et al., 1990) (i) Rim growth: which is found in porous shells. The Si deposition occurs on the rims/edges of the pores which become smaller in diameter during maturation. (ii) Bridge growth, which consists of the production of rod like elements that grow from one node of the cell to another resulting in a more complex skeleton (Anderson, 1983).
(iii) Intermittent growth: polycystines intermittently assimilate siliceous matter within pseudopodia. This Si is quickly transferred to the cytokalimma —which is a cytoplasmic sheath— where it is deposited on the skeleton (Ogane et al., 2014).
For Collodaria and Phaeodaria, the silicification processes are less known; further application of the PDMPO method could solve this problem.

Table of contents :

Introduction 
1. Silica and the marine silicon cycle
2. Rhizaria, conspicuous organisms in the ocean
PhD Objectives
Chapter 1 – Estimating biogenic silica in the global ocean 
Introduction
Material and Methods
Results
Discussion
Chapter 2 – Biogenic silica production of Rhizaria in contrasting environments 
Chapter 2.1. – Rhizaria in the Southern Ocean 
Introduction
Material and methods
Results
Discussion
Chapter 2.2 – Rhizaria in the Atlantic Ocean 
Chapter 3 – Merging imaging technologies and metabarcoding to characterize the Rhizaria community
Introduction
Material and Methods
Results
Discussion
Discussion and perspectives

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