Foraminifera play a crucial role in the global carbon cycle largely due to their prolific biocalcification. Mineralization of shells in Rotaliid Foraminifera have been interpreted to follow two contrasting pathways. The first model assumes in situ mineralization of extracellular organic matrix by attracting calcium and bicarbonate ions that are taken up directly via trans-membrane transport from seawater and/or from the cytosol. The second model assumes that the foraminiferal biocalcification is associated with the production of intracellular amorphous calcium carbonate (ACC), a precursor phase for the formation of calcite shells. However, foraminiferal ACC has not been directly documented. Here, we show evidence of intracellular ACC-bearing vesicles in Rotaliid Foraminifera with a combination of in vivo fluorescence studies, Raman spectroscopy applied to fresh and untouched intracellular vesicles, laboratory synthesis experiments, and chemical/mineralogical analysis of FIB-lamellae from fixed vesicles. We show that foraminiferal ACC vesicles are a natural example of multilamellar vesicles, with a SiO2-core and several layers with differing chemical compositions, likely enabling rapid and efficient ACC production and stabilization. Moreover, we tested and described in detail the first non-destructive autofluorescence method enabling in vivo ACC imaging. This might be applicable to various organisms and increase our understanding of biocalcification throughout the tree of life. STATEMENT OF SIGNIFICANCE: Recognition of Foraminifera calcification process is a precondition to correctly read geochemical parameters archived in biocalcite that are commonly used as reliable proxies for past environmental and climatic conditions, but also to understand carbonate sedimentation that drives the global C cycle. This work presents the key facts to support foraminifera calcification mechanism by true high-Mg amorphous calcium carbonate (Mg-ACC) production within intracellular vesicles. This is a strong argument against commonly existing extracellular foraminiferal (Rotaliida) biomineralisation theory which states that Foraminifera secrete calcium carbonate on an outer organic membrane by attracting Ca2+ and bicarbonate ions that are taken up directly via trans-membrane transport.

Living organisms control the formation of mineral skeletons and other structures through biomineralization. Major phylogenetic groups usually consistently follow a single biomineralization pathway. Foraminifera, which are very efficient marine calcifiers, making a substantial contribution to global carbonate production and global carbon sequestration, are regarded as an exception. This phylum has been commonly thought to follow two contrasting models of either in situ ‘mineralization of extracellular matrix’ attributed to hyaline rotaliid shells, or ‘mineralization within intracellular vesicles’ attributed to porcelaneous miliolid shells. Our previous results on rotaliids along with those on miliolids in this paper question such a wide divergence of biomineralization pathways within the same phylum of Foraminifera. We have found under a high-resolution scanning electron microscopy (SEM) that precipitation of high-Mg calcitic mesocrystals in porcelaneous shells takes place in situ and form a dense, chaotic meshwork of needle-like crystallites. We have not observed calcified needles that already precipitated in the transported vesicles, what challenges the previous model of miliolid mineralization. Hence, Foraminifera probably utilize less divergent calcification pathways, following the recently discovered biomineralization principles. Mesocrystalline chamber walls in both models are therefore most likely created by intravesicular accumulation of pre-formed liquid amorphous mineral phase deposited and crystallized within the extracellular organic matrix enclosed in a biologically controlled privileged space by active pseudopodial structures. Both calcification pathways evolved independently in the Paleozoic and are well conserved in two clades that represent different chamber formation modes.

Living organisms control the formation of mineral skeletons and other structures through biomineralization. Major phylogenetic groups usually consistently follow a single biomineralization pathway. Foraminifera, which are very efficient marine calcifiers, making a substantial contribution to global carbonate production and global carbon sequestration, are regarded as the only exception. This phylum has been commonly thought to follow two contrasting models of either “extracellular in situ matrix mineralization” attributed to hyaline rotaliid shells, or “intracellular vesicle crystallization” attributed to porcelaneous miliolid shells. Our previous results on rotaliids along with those on miliolids in this paper question such a wide divergence of biomineralization pathways within the same phylum of Foraminifera. We found that both groups produced calcareous shells via the intravesicular formation of unstable mineral precursors (Mg-rich amorphous calcium carbonate) supplied by endocytosed seawater and deposited in situ as mesocrystals formed at the site of new wall formation within the organic matrix. We did not observe calcification of the needles within the transported vesicles, which challenges the previous model of miliolid mineralization. Hence, Foraminifera utilize less divergent crystallization pathways, following the recently discovered biomineralization principles. Mesocrystalline chamber walls are therefore created by accumulating and assembling particles of pre-formed liquid amorphous mineral phase within the extracellular organic matrix enclosed in a biologically controlled privileged space by active pseudopodial structures. Both calcification pathways evolved independently in the Paleozoic and are well-conserved in two clades that represent different chamber formation modes.

Sexually produced juvenile scleractinian corals play a key role in the adaptation process of coral reefs, as they are considered to possess an innate plasticity and thus can adjust to changing environmental parameters within a certain range. In this study we investigated in detail the early life stages of the brooding species Leptastrea purpurea to identify, categorize and visualize the critical steps of the complex transformation process from a swimming coral larva to a sessile coral recruit and later to a coral colony. For that, we performed settlement experiments using previously known cues: cycloprodigiosin (CYPRO) and crustose coralline algae (CCA) as well as novel cues: crude extracts of Pseudoalteromonas espejiana and P. piscicida to identify a general, cue-independent settlement pathway. We monitored the development of L. purpurea over 12 months using bright field and fluorescence microscopy. Also we identified the fluorescence signals of L. purpurea with confocal microscopy at four crucial development steps: (A) swimming larva, (B) metamorphosing larva, (C) coral recruit and (D) adult coral. Our methodological approach allowed us to observe an ontogenetic shift of fluorescence signals which provokes the hypothesis that certain fluorescence patterns might be connected to distinct sequential functions in the early life cycle of scleractinian corals. Our observations showed great similarities to the early development of other brooding and spawning corals, making L. purpurea a prospective candidate to be used as a model organism for coral research. Furthermore, our in-depth picture series provides a robust monitoring reference for coral nurseries or field applications and demonstrates the potential of fluorescence as an indicator to instantly determine the growth stage of a developing coral recruit.

The close association between animals and their associated microbiota is usually beneficial for both partners. Here, we used a simple marine model invertebrate, the flatworm Macrostomum lignano , to characterize the host-microbiota interaction in detail. This analysis revealed that the different developmental stages each harbor a specific microbiota. Studies with gnotobiotic animals clarified the physiological significance of the microbiota. While no fitness benefits were mediated by the microbiota when food was freely available, animals with microbiota showed significantly increased fitness with a reduced food supply. The microbiota of M. lignano shows circadian rhythmicity, affecting both the total bacterial load and the behavior of specific taxa. Moreover, the presence of the worm influences the composition of the bacterial consortia in the environment. In summary, the Macrostomum -microbiota system described here can serve as a general model for host-microbe interactions in marine invertebrates.

Calcium carbonate minerals produced by marine organisms play a central role in the global carbon cycle and carbonate sedimentation, which influence the climate by regulating atmospheric CO2 levels. Foraminifera are important marine single-celled organisms that have produced calcite shells for over 300 million years. Here, we present new observations promoting our under-standing for foraminiferal biocalcification by studying Amphistegina lessonii. We integrated in vivo confocal autofluorescence and dye fluorescence imaging with elemental analysis of the cell supporting the concept that the calcite shells of foraminifera are produced via deposition of intracellularly formed Mg-rich amorphous calcium carbonate (Mg-ACC) particles that transform into a stable mineral phase. This process is likely accompanied by the activity of endosymbiotic microalgae and seawater-derived endocytic vesicles that provide calcification substrates such as DIC, Ca2+, and Mg2+. The final transformation of semi-liquid amorphous nanoparticles into a crystalline shell was associated with Mg2+ liberation.

The global degradation of coral reefs is steadily increasing with ongoing climate change. Yet coral larvae settlement, a key mechanism of coral population rejuvenation and recovery, is largely understudied. Here, we show how the lipophilic, settlement-inducing bacterial pigment cycloprodigiosin (CYPRO) is actively harvested and subsequently enriched along the ectoderm of larvae of the scleractinian coral Leptastrea purpura. A light-dependent reaction transforms the CYPRO molecules through photolytic decomposition and provides a constant supply of hydrogen peroxide (H2O2), leading to attachment on the substrate and metamorphosis into a coral recruit. Micromolar concentrations of H2O2 in seawater also resulted in rapid metamorphosis, but without prior larval attachment. We propose that the morphogen CYPRO is responsible for initiating attachment while simultaneously acting as a molecular generator for the comprehensive metamorphosis of pelagic larvae. Ultimately, our approach opens a novel mechanistic dimension to the study of chemical signaling in coral settlement and provides unprecedented insights into the role of infochemicals in cross-kingdom interactions.

Many invertebrate species inhabit coastal areas where loads of plastic debris and microplastics are high. In the current case study, we exemplarily illustrate the principal processes taking place in the Atlantic ditch shrimp, Palaemon varians, upon ingestion of microplastics. In the laboratory, shrimp readily ingested fluorescent polystyrene microbeads of 0.1-9.9 mu m, which could be tracked within the widely translucent body. Ingested food items as well as micro-particles cumulate in the stomach where they are macerated and mixed with digestive enzymes. Inside the stomach, ingested particles are segregated by size by a complex fine-meshed filter system. Liquids and some of the smallest particles (0.1 mu m) pass the filter and enter the midgut gland where resorption of nutrients as well as synthesis and release of digestive enzymes take place. Large particles and most of the small particles are egested with the feces through the hindgut. Small particles, which enter the midgut gland, may interact with the epithelial cells and induce oxidative stress, as indicated by elevated activities of superoxide dismutase and cellular markers of reactive oxygen species. The shrimp indiscriminately ingest microparticles but possess efficient mechanisms to protect their organs from overloading with microplastics and other indigestible particles. These include an efficient sorting mechanism within the stomach and the protection of the midgut gland by the pyloric filter. Formation of detrimental radical oxygen species is counteracted by the induction of enzymatic antioxidants.

Abstract. Deep-sea Cibicidoides pachyderma (forma mundulus) and related Cibicidoides spp. were cultured at in situ pressure for 1-2 days, or 6 weeks to 3 months. During that period, fluorescence analyses following BCECF-AM (2’,7’-bis(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester) or Calcein AM (4,5-Bis((N,N-bis(carboxymethy)amino)methyl)fluorescein acetoxymethylester) labelling, revealed a persisting cytoplasmic sheet or envelope surrounding the Cibicidoides tests. Thus, the Cibicidoides shell can be considered rather as an internal than an external cell structure. A couple of days to a week after being transferred into high-pressure aquaria and adjusted to a pressure of 115 bar, the foraminifera changed from a mobile to a more or less sessile living mode. During this quasi sessile way of life, a series of comparably thick static ectoplasmic structures developed that were not resorbed or remodelled but, except for occasional further growth, remained unchanged throughout the experiments. Three different types of these ‘permanent structures’ were observed: A) Ectoplasmic ‘roots’ were common in adult C. pachyderma, C. lobatulus and C. wuellerstorfi specimens. In our experiments single ectoplasmic ‘roots’ grew to maximum 700 times the individuals shell diameter and were presumably used to anchor the specimen in an environment with strong currents. B) Ectoplasmic ‘trees’ describe rigid ectoplasmic structures directed into the aquarium’s water body and were used by the foraminifera to climb up and down these ectoplasmic structures. Ectoplasmic ‘trees’ were so far only observed in C. pachyderma and enabled the ‘tree’-forming foraminifera to elevate itself above ground. C) Ectoplasmic ‘twigs’ were used to guide and hold the more delicate pseudopodial network when distributed into prevailing currents, and were, in our experiments, also only developed in C. pachyderma specimens. Relocation of a specimen usually required to tear apart and leave behind the rigid ectoplasmic structures, eventually also the envelope surrounding the test. Apparently, these rigid structures could not be resorbed or reused.

Marine sponges harbor many symbionts, which constitute a large amount of the sponge's dry weight. The reef sponges Agelas sp. produce a brominated pyrrole alkaloid, Ageladine A, which accumulates in acidic cellular compartments and has previously been used as a pH sensitive, fluorescent dye. Ageladine A may therefore accumulate in the acidic thylakoid lumen of photosynthetic cells. The ecophysiological role of Ageladine A in the sponge biome, however, is still unresolved. Here we show that Ageladine A possibly acts as an additional light harvesting molecule for photosynthesis in Synechococcus bacillaris, a cyanobacteria similar to the sponge's symbionts. A joint modeling and experimental approach revealed that Ageladine A facilitates photosynthesis of Synechococcus, when the cells are exposed to UV light of 370-380nm wavelength. While Ageladine A absorbs in the UV range, it fluoresces blue, matching the blue absorbance of chlorophyll a. In the presence of Ageladine A, photosynthetic production of O2 increased 2.54- and 3.1-fold, in the experiments and the model, respectively. This study presents a type of symbiosis, in which light is the major currency of species interaction. To verify the applicability of these results to other close relatives of sponge symbionts, two additional strains of Synechococcus sp., and two diatom species, are being tested. The current experiments differ from the initial ones, however, since UV-A radiation of a wider spectrum is used. In addition to these experiments, the applicability of the principle to a wide variety of marine and freshwater algae will be assessed.

Baltic mussels (Mytilus spp.) were exposed to the explosive trinitrotoluene (TNT) for 96 h (0.31-10.0 mg/L) and 21 d (0.31-2.5 mg/L). Bioaccumulation of TNT and its degradation products (2- and 4-ADNT) as well as biological effects ranging from the gene and cellular levels to behaviour were investigated. Although no mortality occurred in the concentration range tested, uptake and metabolism of TNT and responses in antioxidant enzymes and histochemical biomarkers were observed already at the lowest concentrations. The characteristic shell closure behaviour of bivalves at trigger concentrations led to complex exposure patterns and non-linear responses to the exposure concentrations. Conclusively, exposure to TNT exerts biomarker reponses in mussels already at 0.31 mg/L while effects are recorded also after a prolonged exposure although no mortality occurs. Finally, more attention should be paid on shell closure of bivalves in exposure studies since it plays a marked role in definining toxicity threshold levels.

Harmful effects caused by the exposure to paralytic shellfish toxins (PSTs) and bioactive extracellular compounds (BECs) on bivalves are frequently difficult to attribute to one or the other compound group. We evaluate and compare the distinct effects of PSTs extracted from Alexandrium catenella (Alex5) cells and extracellular lytic compounds (LCs) produced by A. tamarense (NX-57-08) on Mytilus edulis hemocytes. We used a 4 h dose–response in vitro approach and analyzed how these effects correlate with those observed in a previous in vivo feeding assay. Both bioactive compounds caused moderated cell death (10–15%), being dose-dependent for PST-exposed hemocytes. PSTs stimulated phagocytic activity at low doses, with a moderate incidence in lysosomal damage (30–50%) at all tested doses. LCs caused a dose-dependent impairment of phagocytic activity (up to 80%) and damage to lysosomal membranes (up to 90%). PSTs and LCs suppressed cellular ROS production and scavenged H2O2 in in vitro assays. Neither PSTs nor LCs affected the mitochondrial membrane potential in hemocytes. In vitro effects of PST extracts on M. edulis hemocytes were consistent with our previous study on in vivo exposure to PST-producing algae, while for LCs, in vivo and in vitro results were not as consistent.

Fluorescence measurements of the marine flatworm Macrostomum lignano were performed during exposure to the explosive TNT and its main derivatives 2-ADNT and 4-ADNT, using calcein AM, the acetoxymethylester of calcein, and the autofluorescence of its food (diatoms). Lethality was found to depend on temperature and exposure time. After 12 days of exposure to a concentration of 33,3 mg/L 2-ADNT and 4-ADNT, the lethality at 30 °C (100%) was strongly increased compared to 21 °C (~60%). First deaths were observed after four days of exposure. Using lower concentrations (≤3,33 mg/L) of all three compounds, the activity of ABC transporters (ATP binding cassette transporter) was determined using calcein as reporter dye. Worms exposed to toxicants for 72 h showed a significant upregulation of ABC transporter activity during exposure to 3,33 mg/L 2-ADNT and 4-ADNT, and 3 mg/L TNT demonstrating the efficacy of this cellular first line defense. A distinct behavioral defense of the worms decreased the uptake of 2-ADNT and 4-ADNT (0,033 mg/L) as they reduced feeding shown by diminished autofluorescence of algae in the gut.

The vulnerability of fish embryos and larvae to environmental factors is often attributed to a lack of adult-like organ systems (gills) and thus insufficient homeostatic capacity. However, experimental data supporting this hypothesis are scarce. Here, by using Atlantic cod (Gadus morhua) as a model, the relationship between embryo vulnerability (to projected ocean acidification and warming) and homeostatic capacity was explored through parallel analyses of stage-specific mortality and in vitro activity and expression of major ion pumps (ATP-Synthase, Na+/K+-ATPase, H+-ATPase) and co-transporters (NBC1, NKCC1). Immunolocalization of these transporters was used to study ionocyte morphology in newly-hatched larvae. Treatment-related embryo mortality until hatch (+20% due to acidification and warming) occurred primarily during an early period (gastrulation) characterized by extremely low ion transport capacities. Thereafter, embryo mortality decreased in parallel with an exponential increase in activity and expression of all investigated ion transporters. Significant changes in transporter activity and expression in response to acidification (+15% activity) and warming (-30% expression) indicate some potential for short-term acclimatization, although likely associated with energetic trade-offs. Interestingly, whole-larvae enzyme capacities (supported by abundant epidermal ionocytes) reached levels similar to those previously measured in gill tissue of adult cod, suggesting that early-life stages without functional gills are better equipped in terms of ion homeostasis than previously thought. This study implies that the gastrulation period represents a critical transition from inherited (maternal) defenses to active homeostatic regulation, which facilitates enhanced resilience of later stages to environmental factors.

Recent advances in fluorescence imaging facilitate actualistic studies of organisms used for palaeoceanographic reconstructions. Observations of cytoskeleton organisation and dynamics in living foraminifera foster understanding of morphogenetic and biomineralisation principles. This paper describes the organisation of a foraminiferal actin cytoskeleton using in vivo staining based on fluorescent SiR-actin. Surprisingly, the most distinctive pattern of SiR-actin staining in foraminifera is the prevalence of SiR-actin-labelled granules (ALGs) within pseudopodial structures. Fluorescent signals obtained from granules dominate over dispersed signals from the actin meshwork. SiR-actin-labelled granules are small (around 1 µm in diameter) actin-rich structures, demonstrating a wide range of motility behaviours, from almost stationarily oscillating around certain points to exhibiting rapid motion. These labelled microstructures are present both in Globothalamea (Amphistegina, Ammonia) and Tubothalamea (Quinqueloculina). They are found to be active in all kinds of pseudopodial ectoplasmic structures, including granuloreticulopodia, globopodia, and lamellipodia, as well as within the endoplasm. Several hypotheses are set up to explain either specific or non-specific actin staining. Two hypotheses regarding their function are proposed if specific actin labelling is taken into account: (1) granules are involved in endocytosis and intracellular transport of different kinds of cargo, or (2) they transport prefabricated and/or recycled actin fibres to the sites where they are needed. These hypotheses are not mutually exclusive. The first hypothesis is based on the presence of similar actin structures in fungi, fungi-like protists, and some plant cells. The later hypothesis is based on the assumption that actin granules are analogous to tubulin paracrystals responsible for efficient transport of tubulin. Actin patches transported in that manner are most likely involved in maintaining shape, rapid reorganisation, and elasticity of pseudopodial structures, as well as in adhesion to the substrate. Finally, our comparative studies suggest that a large proportion of SiR-actin-labelled granules probably represent fibrillar vesicles and elliptical fuzzy-coated vesicles often identified in transmission electron microscope images.

Fluorescent natural compounds have been identified in several marine hosts of microalgae. Their prevalence, and the energy the host is expending on their synthesis, suggests an important, yet poorly understood ecological role. It has been suggested that some of these natural products may enhance the photosynthesis of microbial symbionts. In this study, the effect of Ageladine A (Ag A), a pH-dependent fluorophore found in sponges of the genus Agelas, on the photosynthesis of nine microalgal species and strains was examined. The data showed that the variety of effects of Ag A additions differed between species, and even strains within a species. While in one strain of Synechococcus sp., the presence of Ag A increased gross photosynthesis under UV light exposure, it decreased in another. And while in the chlorophyte T. chuii overall metabolic activity was greatly reduced under all forms of lighting, photosynthesis in T. lutea was positively affected by the addition of Ag A. The variety of effects of Ag A on photosynthesis observed in this study indicate a complex interaction of Ag A with microalgal cells and suggests that a host may be able to shape its own symbiotic microbiome with self-produced natural products.

The contamination of coastal regions with different toxicants, including heavy metal ions such as copper and cadmium jeopardize health and survival of organisms exposed to this habitat. To study the effects of high copper and cadmium concentrations in these marine environments, we used the flatworm Macrostomum lignano as a model. This platyhelminth lives in shallow coastal water and is exposed to high concentrations of all toxicants that accumulate in these sea floors. We could show that both, cadmium and copper show toxicity at higher concentrations, with copper being more toxic than cadmium. At concentrations below acute toxicity, a reduced long-term survival was observed for both metal ions. The effects of sublethal doses comprise reduced physical activities, an increase in ROS levels within the worms, and alterations of the mitochondrial biology. Moreover, cell death events were substantially increased in response to sublethal concentrations of both metal ions and stem cell activity was reduced following exposure to higher cadmium concentrations. Finally, the expression of several genes involved in xenobiotic metabolism was substantially altered by this intervention. Taken together, M. lignano has been identified as a suitable model for marine toxicological studies as it allows to quantify several relevant life-history traits as well as of physiological and behavioral read-outs.

In several marine hosts of microalgae, fluorescent natural products may play an important role. While the ecological function of these compounds is not well understood, an interaction of these molecules with the photosynthesis of the symbionts has been suggested. In this study, the effect of Ageladine A (Ag A), a pH-dependent fluorophore found in sponges of the genus Agelas, on microalgal fluorescence was examined. The spectra showed an accumulation of Ag A within the cells, but with variable impacts on fluorescence. While in two Synechococcus strains, fluorescence of phycoerythrin increased significantly, the fluorescence of other Synechococcus strains was not affected. In four out of the five eukaryote species examined, chlorophyll a (Chl a) fluorescence intensity was modulated. In Tisochrysis lutea, for example, the position of the fluorescence emission maximum of Chl a was shifted. The variety of these effects of Ag A on microalgal fluorescence suggests that fluorophores derived from animals could play a crucial role in shaping the composition of marine host/symbiont systems.