Molybdenum is an essential micronutrient, but because of its toxicity at high concentrations, its accumulation in living organisms has not been widely demonstrated. In this study, we report that the marine sponge Theonella conica accumulates exceptionally high levels of molybdenum (46,793 micrograms per gram of dry weight) in a wide geographic distribution from the northern Red Sea to the reefs of Zanzibar, Indian Ocean. The element is found in various sponge body fractions and correlates to selenium. We further investigated the microbial composition of the sponge and compared it to its more studied congener, Theonella swinhoei. Our analysis illuminates the symbiotic bacterium Entotheonella sp. and its role in molybdenum accumulation. Through microscopic and analytical methods, we provide evidence of intracellular spheres within Entotheonella sp. that exhibit high molybdenum content, further unraveling the intricate mechanisms behind molybdenum accumulation in this sponge species and its significance in the broader context of molybdenum biogeochemical cycling.
Marine sponge Theonella conica and its symbiotic bacterium Entotheonella sp. accumulate high levels of the element molybdenum.
INTRODUCTIONSponges (phylum Porifera) are found in all types of marine environments and contribute to global carbon (C), nitrogen (N), and silicon (Si) cycles (1–3). They filter-feed by processing up to 50,000 times their body volume of seawater daily (4). Sponges are essential to the marine benthos ecosystem and can serve as biomonitors for environmental trace elements. They are ubiquitous, prevalent in certain ecosystems, have a long lifespan, are readily collected, sessile filter-feeders, and exhibit high tolerance to various pollutants (3, 5, 6). The high volume of seawater passing through sponges exposes them to many toxic elements, even if present at trace concentrations, which can accrue to a large amount (7). Previous research has shown that different sponge species can accumulate trace elements (7, 8), which vary on the basis of the species and environmental conditions. Some sponge species are highly influenced by environmental element concentration, while others exhibit biological control of their element concentration (7). Substantial amounts of trace elements in sponges indicate they either have a high tolerance toward various chemical substances or have developed detoxification mechanisms (9). Sponges were recorded to accumulate higher trace element concentrations, notably arsenic (As) and molybdenum (Mo), than other organisms known to accumulate trace elements, such as bivalves and seaweed (3, 7, 10, 11). All trace elements are toxic above a certain bioavailable (water-soluble) concentration (12). The thresholds vary by species, depending on their detoxification mechanisms.
A study on the accumulation of elements in sponges from the Gulf of Aqaba (Red Sea) found that Theonella swinhoei (order Tetractinellida) actively coaccumulates large amounts of the toxic elements arsenic (As) and barium (Ba) (7, 13, 14). Previously, we published that the sponge-associated filamentous bacterium Entotheonella sp. drives the accumulation of arsenic and barium by mineralizing them inside its cells (14). Theonella conica is congeneric to T. swinhoei (Fig. 1A). Both Theonella spp. sponges are distributed along the Indian Ocean (T. swinhoei is also present in the Pacific Ocean); however, T. conica is much less common in the northern Gulf of Aqaba and relatively less studied, and studies have been focused on the sponge’s ability to produce bioactive compound, but not much is known of its relation with microbial organisms (15–18). In addition, while, morphologically, the exterior of the two sponges is quite similar, the interior of T. conica is dark blue (Fig. 1B) compared to the cream color of T. swinhoei interior (18). Since elements accumulation in T. swinhoei is biologically mediated, we hypothesized that a similar phenomenon occurs in T. conica. The distinct blue interior of T. conica could be attributed to molybdenum compounds, potentially in the form of molybdenum blue, which is known for imparting a characteristic coloration. This hypothesis was supported by preliminary observations where, upon fixation of a fresh T. conica specimen from the Red Sea in formalin, there was a notable leaching of the blue coloration into the solvent. The solvent subsequently exhibited a high molybdenum concentration of 44,765 μg/ml. Although these preliminary findings did not allow us to definitively confirm the presence of molybdenum blue due to the limited scope of the sample and the absence of detailed quantitative analysis, they did indicated that a substantial amount of molybdenum is released into the fixative, suggesting a high concentration of molybdenum compounds within the sponge.
Fig. 1. T. conica from the Red Sea.(A) The sponge seen in the Gulf of Aqaba at 30 m in depth before sampling. (B) Image showing the sponge’s external maroon (ectosome) and interior blue (endosome) parts following sample removal.
Molybdenum is a transition metal and an essential micronutrient. Similar to other trace elements, it is toxic above a certain water-soluble concentration (12, 19, 20). The element is considered a pollutant in water and soil due to its use in industries such as byproducts in copper mining. Naturally, molybdenum has a high affinity for many oxide minerals, particularly aluminum, iron, and manganese. That is why large quantities of molybdenum are often found in sedimentary deposits rich in oxides, such as hydrothermal manganese crusts (21). In addition, metal molybdates can form naturally and are known for their stability in solid and liquid phases, such as CaMoO4 (powellite) (22). In open seawater, molybdenum is relatively abundant, with a concentration of 10 μg of Mo/liter, due to the prevalence and low reactivity of the molybdate ion (MoO42−) (23, 24), which is influenced by geological processes in the oceanic cycle (25). Molybdenum has been identified in various invertebrates, including sponges, at concentrations of up to 118 μg/g of dry weight (11, 26). Molybdenum does not exhibit biological activity independently, but it is an active cofactor in over 60 enzymes, such as nitrogenase and nitrate reductase. These enzymes facilitate nitrogen fixation and nitrate reduction reactions, respectively (27, 28). In marine environments, rivers serve as the primary source of molybdenum (12). Approximately half of this input is eliminated through deep-sea deposits, with the remainder presumed to be removed in near-shore reducing sediments. Unexpectedly, no correlation was found between the proximity of sponges to outfall and the measured molybdenum concentration, for example, in the sponge Halichondria phakellioides (11). This might suggest that the abundance of metal-binding bacteria within the sponge might influence molybdenum uptake more substantially than the element’s availability in the surrounding environment (11). Several bacteria were found to precipitate molybdenum in various bacterial systems, including microbial mats, metal-contaminated soil, and marine sponges (29, 30). In T. swinhoei, the precipitation of metals is influenced by its symbiotic bacteria, and in the case of T. conica, it is unclear which (if any) bacterium is responsible for its high concentration of molybdenum.
In this work, we explored the elemental composition of T. conica across a wide geographic distribution and demonstrated that it is a hyperaccumulator of molybdenum metal. Microscopic and analytic methods were used to analyze the composition and location of elements inside the sponge body and their relation to different sponge cell fractions. We further investigated the microbial community structure of T. conica and the role of its dominant bacterium, Entotheonella sp., in the process of molybdenum hyperaccumulation.
RESULTS Elements accumulation in T. conicaElements accumulation by T. conica was initially examined in freeze-dried sponge samples (n = 4) collected from Zanzibar (6°6′ N, 39°9′ E) and the northern Red Sea (n = 4). To cover a wide range of possibly accumulated elements, we measured 22 elements by inductively coupled plasma mass spectrometry (ICP-MS) in the Zanzibar samples and ICP atomic emission spectroscopy (AES) in the Red Sea samples (Fig. 2A).
Fig. 2. Elements concentration (micrograms per gram of dry weight) in T. conica (means ± SE).(A) The mean concentration of elements in total sponge tissue (n = 8) from two distinct geographic locations, Zanzibar and the northern Red Sea. Elements are ordered in an alphabetic order. (B) Concentration of elements in four enriched sponge fractions (n = 4) divided into the sponge’s outer layer—the ectosome (Ecto), Entotheonella sp. (Ento), sponge cells (SPC), and unicellular bacteria (UniB). Both sodium (Na) and sulfur (S) were excluded from the figures for their high concentration in the samples and marine environment in general.
Of all elements measured, molybdenum stood out, with not only an average concentration of 46,793 (±10,110 SE) μg/g of dry weight in the Red Sea sponge samples (Fig. 2A) but also high (28,423 ± 4451 SE) in the Zanzibar samples. Molybdenum concentration was tested for a difference between sites by a permutation two-sample t test, followed by the Wilcoxon rank sum exact test, which did not find a significant difference between sites concerning molybdenum (probably because of the small sampling pole). The analysis revealed that selenium concentration in the Red Sea samples was extremely higher (average of 11,292.8 μg/g) than in the Zanzibar sponges (~20 μg/g; P = 0.014).
Sponges do not have defined tissues or organs. However, there is a possibility that elements are stored in separate areas of the sponge body or within different types of cells responsible for that accumulation (sponge cells and bacteria). Following the initial discovery of high molybdenum concentrations in T. conica and to locate where the sponge accumulates 10 elements of interest, different enriched cellular fractions of fresh Red Sea samples were examined (Fig. 2B). The highest molybdenum concentration was measured in the bacterium Entotheonella sp. and in the ectosome fractions (average of 30,732 and 33,616 μg/g, respectively). Selenium had a similar correlation in those fractions as well. Furthermore, in the Entotheonella sp. fraction, molybdenum was the highest measured element of all tested elements. A post hoc analysis grouped molybdenum with the abundant marine environments macronutrients calcium (Mo-Ca = 0, Stat = −1.386, P = 0.1656, P.adj = 0.2127) and potassium (K-Mo = 0, Stat = −2.53, P = 0.0897, P.adj = 0.152). The grouping of molybdenum with the two elements was based on no significant difference in element concentration in the Entotheonella sp. fraction.
In vivo localization of molybdenumTo examine the distribution of molybdenum in the sponge body, we made the scanning electron microscopy analysis with energy-dispersive spectroscopy (SEM-EDS) measurements (Fig. 3A), including the ectosome and endosome (outer and inner parts) of the sponge. The corresponding peaks are shown in Fig. 3B. The major molybdenum peak at ~2.2 kV was indistinguishable from the sulfur (S) peak; however, molybdenum is threefold denser than sulfur (9.01 compared to 2.07 kg/m3), and using a backscattered electron detector, objects with higher density are seen brighter in the SEM imaging. We focused the ion beam on brighter objects to locate molybdenum-containing minerals such as CaMoO2, MoO2, and MoS2, which are known to precipitate in aquatic environments (24, 31). A close look at the sponge’s ectosome (Fig. 3, A and B, and fig. S1) revealed a high density of spherical-shaped granules. Spectrum EDS mapping indicated high levels of molybdenum (27.75 weight %) as well as calcium, silica, oxygen, and carbon in these spheres (fig. S1), while the adjacent areas did not. In the Zanzibar samples (fig. S2), molybdenum and calcium in granules (10 granules from three sponges) accounted for 32.2% (±3.7 SE) and 14.5% (±2.2 SE) of the total weight of the granules, respectively. An example of such a granule and its corresponding peaks is presented in Fig. 4. A similar composition was found in the Red Sea samples (three granules from two sponges), with the weight % of molybdenum and calcium being 32.3% (±1.5 SE) and 12.4% (±0.98 SE), respectively. The molybdenum:calcium atomic ratio was 1.01 (±0.047 SE, n = 13). This ratio fits best with calcium molybdate (powellite, CaMoO4), a mineral with low solubility (22, 24).
Fig. 3. T. conica ectosome contains molybdenum-enriched spherical granules.SEM micrographs of T. conica showing molybdenum precipitation. Images were taken with backscatter detection mode. (A) An enlargement of the ectosome section reveals a high density of epibionts. Green arrows point to two diatom morphologies (algae with silica cell walls). The yellow arrow points to an unidentified spherical object (8 ± 2 μm), and the orange arrow points to the sponge’s ectosome layer. (B) EDS spectrum comparison of the spherical objects and the sponge’s ectosome.
Fig. 4. Molybdenum- and calcium-enriched granules in the endosome of T. conica.(A) SEM micrographs of T. conica endosome showing molybdenum and calcium precipitation as spherical aggregates. Images were taken with backscatter detection mode. The cross marks the area analyzed (spectrum 1). Insert (top left corner) provides relative values (weight %) of detected elements by EDS. (B) EDS of spectrum 1 with its corresponding peaks of molybdenum (Mo) and calcium (Ca).
Focusing on the sponge’s endosome, we identified a high abundance of Entotheonella sp. morphologically (fig. S3). This filamentous bacterium did not reflect any molybdenum mineral on its cell membranes, yet molybdenum may be precipitated intracellularly. That can be verified and presented in transmission electron microscopy (TEM)–EDS of an isolated Entotheonella fraction.
Entotheonella sp. precipitated molybdenum intracellularlyIn T. swinhoei, Entotheonella was found to mineralize both arsenic and barium inside its cell (14). Therefore, we looked to this bacterium for a similar mechanism to explain T. conica’s high molybdenum concentrations.
Following the initial results of high molybdenum concentrations in the enriched Entotheonella fraction from T. conica (Fig. 2B), we examined viable bacteria under light microscopy (×100). The bacteria cells contained intracellular spheres exhibiting a distinguished blue hue, while some cells were partly yellow (Fig. 5A). The different colors seen in the bacteria cells could result from molybdenum compounds, which exhibit various colors depending on their chemical composition. Some standard colors associated with molybdenum compounds include sodium molybdate (Na2MoO4), which forms yellow solutions or crystals, and certain molybdenum oxides, such as molybdenum trioxide (MoO3), can appear blue in their solid form (24).
Fig. 5. Intracellular accumulation of molybdenum in Entotheonella sp.(A) Light microscopy imaging (×100) of isolated bacterium filaments with blue vacuoles. (B) TEM micrographs of thin sections of Entotheonella sp. Blue arrows mark the high-density singular sphere in each cell. (C) EDS spectrum of the chosen area from one high-density sphere (shown in blue and black images). Black arrows point to major and minor molybdenum peaks at ~2.2 and 17 kV.
Fixed bacterium filaments were examined under TEM with an EDS module to locate the presence of molybdenum inside the cells. Each cell of the immobilized bacterium (n = 11) contained high-density spheres in heterogeneous sizes and numbers (Fig. 5B). EDS spectrum confirmed the presence of molybdenum in the dense part of each sphere (Fig. 5C) and measured six dominant elements for an atomic fraction (Fig. 6) and atomic mass (%). The molybdenum was measured at 14.2 ± 2.9 atomic fraction and 13.3 ± 3 atomic mass (% ± SE). Other dominant elements found in the spheres were oxygen, carbon, calcium, sulfur, and iron. During the analysis, fast Fourier transform (FFT) was applied on the dense part of the spheres to detect crystalline minerals, yet no signal was retained.
Fig. 6. Atomic fraction (%) of elements of unidentified spheres in Entotheonella sp.(A) Measurements of six dominant elements found in the spheres (n = 11) inside the bacterium cells. The graph shows the atomic fraction (%) ± SE. (B) One area of spheres is shown by the TEM-EDS module with the identification of three elements: Molybdenum (Mo), iron (Fe), and sulfur (S). HAADF, high-angle annular dark-field.
X-ray diffraction for mineral identificationSince no specific molybdenum-containing compound or mineral could accurately be identified by TEM-EDS, probably due to high organic matter and poor crystallinity of the freeze-dried Entotheonella sp. cells, we used synchrotron high-resolution powder x-ray diffraction (HRPXRD) to study the presence of crystalline phases, which could explain the high molybdenum concentration within the sponge. HRPXRD analyses were performed on the freeze-dried samples of five fractions (Entotheonella, unicellular bacteria, sponge cells, ectosome, and total sponge). Collected diffraction patterns revealed the presence of crystalline molybdenum oxide (MoO2) in ectosome, Entotheonella, and sponge cells fractions. Unicellular bacteria fraction and the total sponge sample contained a major contamination phase of NaCl, which disabled further quantitative analysis. Qualitatively, the crystalline phases identified in the samples were sodium chloride (NaCl), calcium magnesium carbonate (CaMgCaCo3), potassium sodium chloride (KNaCl), aluminum selenate (Al2Se3), and molybdenum oxide (MoO2) (fig. S4).
Analysis of T. conica’s microbial communityExamining the endosome of T. conica (fig. S3) using SEM revealed a dense community of unicellular microorganisms in addition to filamentous (identified as Entotheonella sp.) bacteria (32), which were very abundant, similar to the Entotheonella sp. present in T. swinhoei. Since T. swinhoei is also a hyperaccumulator of metals, we were motivated to compare the microbial composition of the two closely related sponges. The analysis of the microbial community structure in three T. conica samples resulted in the grouping of 5456 ASVs (amplicon sequence variants; genus level > 97% similarity) that were identified to the lowest taxonomic level possible (species), before grouping to 726 operational taxonomic units (OTUs). Of these OTUs, 12% were also found in the congener T. swinhoei. A total of 1,216,270 16S ribosomal RNA (rRNA) V4 region amplicon reads were successfully recovered from T. conica (n = 3) and T. swinhoei samples (n = 5), with an average of 405,423 ± 69,308 SE reads per sample. After noise reduction (filtering, denoising, and chimera removal), 340,153 reads were retained, averaging 113,384 ± 4248 SE reads per sample. ASVs assigned as chloroplasts, mitochondria, or eukaryotes were removed, leaving 1003 ASVs characterized as belonging to 41 bacterial and archaeal phyla (Fig. 7). In T. conica, 11 ASVs belonged to the phylum Entotheonellaeota, the 18th most dominant in those samples and comprising >10% of the total abundance. Proteobacteria was the most dominant phylum, comprising 52% of the ASVs’ relative abundance. Negative control samples yielded 87 reads belonging to phyla Firmicutes and Proteobacteria.
Fig. 7. Relative abundance of microbial communities in T. conica and T. swinhoei samples (n = 3 and 5, respectively).The left column presents negative control. The figure presents the abundance of the various phyla in each sample.
Further nonmetric multidimensional scaling analysis revealed significant differences in the community structure of associated bacteria between the two Theonella species. The species identity explained 67% of the observed variation in the core microbiome [permutational multivariate analysis of variance (PERMANOVA) test, F = 8.76, P = 0.02, R2 = 66.67; Fig. 8]. The main phyla that were responsible for the difference between the two groups are Firmicutes and Fusobacteriota, which were absent from T. swinhoei samples. By looking at the family level of the sequenced OTUs from the two Theonella spp. (table S1), we count 30 unidentified families in T. conica compared to 19 in T. swinhoei (17 shared between the sponge species).
Fig. 8. Nonmetric multidimensional scaling plot.The core microbial community composition of T. conica (TC; blue), T. swinhoei (TS; green), and negative control (red). Stress = 0.005, R2 = 0.66, and k = 2 (PERMANOVA test, F = 8.76, P = 0.02).
The 16S rRNA V4 region sequenced from Entotheonella sp. from T. conica is 99.58% identical to the sequence of a previously isolated clone from the Japanese sponge Topsentia sp., which is publicly available in GenBank (accession no. KF926808.1) (33). However, Entotheonella sp. from T. conica is less than 97% identical to Entotheonella sp. from the Red Sea T. swinhoei. No public data are available on T. conica’s microbiome from other geographic locations.
DISCUSSIONThe sponges analyzed in this study were collected over two decades (2001–2021) from two bodies of water (West Indian Ocean and Red Sea), with sampling sites separated as far as 4000 km by direct line or ~5800 km by sea. Despite the vast geographical distance, all T. conica sponges contained high molybdenum concentrations. Three main rivers flow into the Zanzibar channel: the Pangani River in the north, the Wami River in the west, and the Rufiji River in the south, while no major river flows into the Red Sea. There is nonetheless no significant difference in seawater molybdenum concentration between the Red Sea and Zanzibar bodies of water (23), negating a possible effect of river input on sponge accumulation of the element.
It has been commonly assumed that variations in materials and bioactive compounds found in sponges of the same species across a wide geographic range are primarily attributed to environmental conditions. Developmental stage or physiological differences within a species, such as stress or seasonal factors, were also reported (10, 34). In the present study, all samples exhibited molybdenum accumulation in high concentrations (Fig. 2A), suggesting that other (unknown) environmental or biological factors play a role in the accumulation of this element in sponges. Of all the elements measured, molybdenum stood out, with a measured concentration of up to 46,793 μg/g of dry weight. This value is much higher than expected for a micronutrient alone. We assume it to be the highest recorded molybdenum concentration in invertebrates, which were measured to no more than 62 μg/g (10, 11), and even suppressing the concentration measured in bioaccumulating plants in a molybdenum mine (1979 μg/g) (35).
Unexpectedly, the Red Sea T. conica samples recorded high selenium levels, unlike those from Zanzibar. Selenium is a trace element not dominant in seawater or sediment (36). However, it has been previously shown that of 16 Demospongiae sponge species of the Red Sea, 14 actively uptake selenium in higher concentrations than near sediment (7). T. swinhoei exhibited the highest concentration of selenium and almost twice the amount found in all other sponge species tested. Selenium uptake in sponges was attributed to its associated bacteria, which incorporate the element in various proteins (7, 37) Here, the presence of high selenium concentration was observed in all T. conica fractions. The latter was also evident for the high concentration of molybdenum that was not restricted to a particular region of the sponge. The high concentration found in both Entotheonella sp. and ectosome fractions (Fig. 1B), indicating a possible correlation between the accumulation of molybdenum and selenium in sponges. However, this correlation was found only in Red Sea samples, while Zanzibar samples exhibited no significant selenium concentration. One example of a potential codependence mechanism can be found in the dissimilatory enzyme selenate reductase. This molybdenum-dependent membrane-bound enzyme is found in bacteria and is associated with cytoplasmic membrane activity enhanced by molybdenum. The function of the enzyme may be linked to protection against high levels of both selenate and selenite rather than supporting the anaerobic growth of the bacteria (38). Therefore, it was expected to find 37 ASVs and 27,507 reads belonging to the order Veillonellales-Selenomonadales (phylum Firmicutes) in T. conica samples, while none were detected in T. swinhoei. These bacteria, known for their selenium-reducing capabilities, include species such as Megaspheara, notably prevalent in wastewater rich in selenium (39). In addition, Firmicutes are known to harbor a notable number of organisms that exhibit all three traits for selenium utilization and use a wide array of selenoproteins (40). In our study, we found that Firmicutes were one of the main phyla that were responsible for the high difference in microbial composition between the two Theonella sponges (Fig. 8). Selenium toxicity results from its ability to replace sulfur in proteins, causing them to lose their correct folding (41). The existence of aluminum selenate (Al2Se3) mineral in the sponge can be explained by the high affinity of selenium to aluminum oxides (42, 43).
T. conica exhibited high concentrations of molybdenum in its ectosome, where many unidentified spherical-shaped granules were detected. Following the ICP-AES and SEM-EDS results (Figs. 2A, 3, and 4), we assume that those granules contain some form of molybdenum-bearing mineral, most likely calcium molybdate (powellite, CaMoO4), which can form spherical microparticles (44, 45). However, no such mineral was detected by XRD analysis, perhaps because of the high sodium chloride (NaCl) contamination in the different sponge fractions. The XRD results suggest the presence of calcium magnesium carbonate (CaMgCaCo3), which could explain the high calcium concentration levels observed. However, both calcium concentrations measured by ICP-AES and its atomic weight extracted by SEM-EDS were significantly higher (more than fivefold) than those of magnesium. They were also the highest in the ectosome fraction of the sponge, where the calcium- and molybdenum-bearing spherical-shaped granules appear. Calcium molybdate formation depends on the residence time of the water and if its molybdenum and calcium enrichments are sufficient for precipitation. Under natural pH and supersaturated conditions of molybdenum and calcium ions, it can form as coatings on carbonates and silicate minerals (22). These minerals (SiO2 and CaCO3) are abundant in sponges as spicules (sponge skeleton). Therefore, we assume that the process of calcium molybdate mineralization in the sponge could be spontaneous because of the natural conditions in the microenvironment, as mentioned above.
The T. conica blue interior color (Fig. 1B) is most likely due to the presence of molybdenum-blue, which results from molybdate reducing bacteria activity (46). Members of the genus Acinetobacter, which, according to our findings, are represented in T. conica by 40 ASVs (but are absent from T. swinhoei), have been reported to reduce hexavalent molybdenum to form molybdenum-blue under anoxic conditions (47). These anoxic conditions were detected in sponges (48, 49). Another group of bacteria known to precipitate molybdenum is from the genus Desulfovibrio (50), which was also present only in T. conica samples but with fewer ASVs. The large quantity of unidentified families (NAs, not available) in T. conica indicates a vast, yet-to-be-find diversity of bacteria, suggesting numerous potential implications that remain to be uncovered.
Our findings support the existence of biologically induced mineralization accompanied by active accumulation of molybdenum in this sponge, processes led by its symbiont Entotheonella sp.; although it is the 18th most abundant symbiont in the sponge (Fig. 7), it was shown to contain higher levels of the measured molybdenum concentration compared to the unicellular bacteria fraction (Fig. 2B). These findings are particularly notable since previous studies have suggested that Entotheonella has the capability to mineralize other elements, such as arsenic and barium, within its cells (14). Entotheonella’s genomic features indicate a mixotrophic metabolism that is based on sulfate reduction, aerobic and anaerobic respiration, and denitrification (51), implying the importance of molybdenum for the bacterium, since nitrate reductase enzyme reaction is molybdenum dependent (52, 53). Identifying additional dominant elements, including calcium, sulfur, and iron, on intracellular spheres (Figs. 5 and 6) suggests that they may be involved in mineralization processes within this bacterium. The presence of sulfide species in anoxic environments is closely associated with the geochemical behavior of molybdenum in the oceans. Under these conditions, iron availability may play a crucial role in scavenging molybdenum (31). It is reasonable to assume that coprecipitation or absorbance of Mo-polysulfide species on minerals such as FeS2 or FeS occurs in Entotheonella sp. via the reduction of thiomolybdates. The lack of signal retained from applying FFT on the dense part of the intracellular spheres may indicate that those minerals exhibit a disordered or amorphous structure. XRD analysis provided additional evidence of molybdenum-bearing minerals within the bacterium in the form of molybdenum oxide (MoO2). These characteristics raise the possibility that the T. conica–associated Entotheonella sp. plays a similar role to those residing in the congeneric T. swinhoei.
Entotheonella sp. has been coined as a “talented producer” of bioactive compounds (54) since it synthesizes almost all known compounds derived from T. swinhoei (51). Previously, our group hypothesized that in T. swinhoei, this bacterium also acts as a detoxification “organ” of the hosting sponge by mineralizing arsenic and, thus, lowering its toxic effect (14). Here, we believe that a similar phenomenon applies to Entotheonella sp. of T. conica, which mineralizes molybdenum. Sequence examination of the Entotheonella sp. from T. conica reveals it to be closely related to the one from the sponge Topsentia (Demospongiae; Halichondrida). In addition, Topsentia is known for its prolific bioactive compounds production (55–57). Furthermore, T. conica’s Entotheonella was not identical to that of T. swinhoei despite the similarity in elements accumulation in both species. The findings of this study further illuminate the unique capabilities displayed by Entotheonella sp. and highlight the potential for additional research on its role in the molybdenum cycle and implications in biomineralization processes.
MATERIALS AND METHODS SamplingT. conica samples were collected by scuba diving or free diving at 0 to 30 m in depth. Red Sea samples were collected in the Gulf of Aqaba (n = 3) (permit no. 2020/42658; Israel Nature and Parks Authority) during 2020–2021. The Zanzibar sampling site was in the coral reefs of Changu Island, and samples were taken (with permits, ref: AV1/B/VOL IV) in 2001, 2006, and 2013 (n = 4). Shortly after collection, Zanzibar specimens were frozen (−20°C), followed by freeze-drying in Israel. Fresh Red Sea samples were treated for various analyses listed below in addition to freeze-drying for ICP-AES.
Quantitative element analysisQuantification of element concentration was performed for the Zanzibar samples using ICP-MS at the Institute of Earth Sciences, Hebrew University of Jerusalem. Red Sea sample element quantification was achieved using ICP-AES at the Water Research Center, Porter School for Environment and Earth Science, Tel Aviv University. The analytical method selection was determined by the availability of infrastructure in different years, with ICP-MS being used in 2014 and ICP-AES in 2020. This discrepancy in timing should not affect the comparability of the two methods. However, it highlighted a specific issue where sulfur concentration presented interference in the ICP-AES analysis, leading to its complete exclusion from the analysis conducted by both methods. Samples were scanned for the following elements: Ag, Al, As, B, Ba, Ca, Cd, Co, Cr, Cu, Fe, K, Li, Mg, Mn, Mo, Na, Ni, P, Pb, S, Se, Sr, V, and Zn. Red Sea samples were measured for element quantification in different fractions: total sponge tissue, sponge cells, unicellular bacteria, Entotheonella sp. enriched, and sponge’s ectosome (which contains symbiotic cyanobacteria). The sponge separation into fractions was conducted as described by Keren et al. (14) and is based on well-established differential centrifugation protocols (33, 58). Dried sponge material from each sample (100 mg) was acid-digested, following the Environmental Protection Agency method 3050B11. Briefly, samples were heated in concentrated nitric acid (HNO3) to 95°C for 15 min in a hot-block apparatus. Once cooled, additional HNO3 was added, and the samples were reheated to 95°C for 30 min, during which the formation of brown fumes was observed. This step was repeated until no fume formation was detected and ended after 2-hour incubation at 95°C. Once acidic digestion was completed, hydrogen peroxide (H2O2; 30%) was added to the solution in aliquots, and gassing was monitored until it subsided to a minimum or the sample appearance remained unchanged. An additional 2-hour incubation period was allowed to reduce solution volume before cooling to room temperature. Before analysis, samples were diluted in double-distilled H2O and filtered to remove particulate matter.
In vivo element localizationSponge specimens were preserved in a 2.5% glutaraldehyde solution within 0.1 M sodium cacodylate buffer (pH 7.4) for a duration of 24 hours at ambient temperature. Following fixation, the samples underwent dehydration through a graduated series of ethanol concentrations, were dried to the critical point, cut into sections measuring 20 mm, affixed to aluminum stubs, and then sputter-coated with a 10-nm layer of carbon (59). Imaging and elemental analysis were performed using an SEM for morphological analysis, equipped with an EDS for bulk compositional determination. SEM images were captured at different acceleration voltages with the help of an Evehart Thornley secondary electron detector (SE), enabling investigation of the surface topography, and an energy-selective backscattered detector suitable for obtaining a clear compositional contrast (Z-contrast). EDS spectra were collected on points of interest using the accelerating voltage of 10 keV at a working distance of 6 mm. Quantitative analysis was performed using the conventional correction procedure included in INCA software. The final results were normalized to 100% and presented as a relative ratio of elements’ mass (weight %).
Entotheonella sp. cells elemental analysisViable bacterium cells were separated from fresh T. conica tissue by a well-established cell separation procedure (32, 47) and checked for purity of separated fractions under a light microscope. Cells were fixed by high-pressure freezing, followed by freeze-substitution and the addition of uranyl acetate to improve image contrast. A 200-nm slice samples were viewed under TEM/scanning TEM coupled with EDS modules (Titan Themis G2 300, FEI/Thermo Fisher Scientific, the Electron Microscopy Center, Department of Materials Science and Engineering, Technion). Scanning was done at 60 kV at backscattered electron mode to detect localities of electron-dense material, and EDS element analysis was conducted on points of interest. Results of the latter analysis are given in relative ratio of elements atoms (atom %) for extraction of element composition, atomic spectrum, and an atomic fraction (%).
Phase analysis on separated sponge fractionsFreeze-dried powder of each of the five fractions was used for XRD analysis. HRPXRD experiments were carried out using a synchrotron source (beamline ID22, European Synchrotron Radiation Facility, Grenoble, France). Each powder sample was finely grounded and transferred into a borosilicate capillary (0.5 to 0.7 mm in diameter). The capillary was sealed by wax and mounted on a holder. Diffraction patterns were collected using radiation with a wavelength of 0.3542 Å, measured at room temperature.
Construction of microbial communityGenomic DNA was extracted from T. conica and T. swinhoei (n = 3 and 5, respectively) using a DNeasy PowerSoil kit (QIAGEN) following the manufacturer’s protocol, with minor modifications: Sponge samples were chopped to 1-mm pieces with a sterile scalpel. The pieces were added to the PowerBead Pro Tubes, vortexed for 3 min, and placed in a preheated bath at 60°C, followed by the manufacturer’s protocol. Raw DNA samples were sent for the preparation and sequencing of the V4 of the 16S rRNA gene libraries, using primer pair 515F and 806R. The libraries were then sequenced using MiSeq v2 Kit (500 cycles) to generate 2× 300–base pair reads via Illumina MiSeq (Illumina Inc., San Diego, CA, USA) at Mr.DNA (Texas, USA). Raw FastQ files of the reads were processed for quality filtering, merging pairs of reads, and assigned taxonomic inference using the DADA2 package (60) following its online pipeline tutorial v1.12 and using SILVA v138.1 (61) for rRNA reference database of the classification of the sequence to ASVs (62). The molecular identity of the dominant bacterium Entotheonella sp. was established of a fresh sample of isolated bacteria preserved in RNAlater Stabilization Solution (Thermo Fisher Scientific), in a ratio of 1:3 with calcium-magnesium free seawater (CMF-SW) and frozen at −80°C until further use. Using an established protocol by Wilson et al. (33), the 16S rRNA section was amplified with primers 1492R and 27F, followed by 1290R and 271F in a hot start nested polymerase chain reaction (33). Products were cleaned with EXO nuclease to remove excess primers and Sanger-sequenced using an ABI 3500xl genetic analyzer (TAU, Israel). The resulting 16S rRNA gene sequences were identified by BLAST searches of the National Center for Biotechnology Information database for sequence similarity analysis.
Statistical analysisQuantitative data were analyzed using R statistical software, including element concentrations from ICP-MS and ICP-AES and microbial community structures from 16S rRNA gene sequencing. Element concentrations across different locations (Red Sea versus Zanzibar) and sponge fractions were compared using one-way ANOVA with Tukey’s post hoc test for post hoc analysis or Kruskal-Wallis test for nonparametric data, following log transformation to meet normality and homogeneity of variance prerequisites. Microbial community diversity (α-diversity, measured by Shannon index and observed species richness, and β-diversity, assessed through Bray-Curtis dissimilarity and visualized via nMDS) and composition differences were evaluated using PERMANOVA. All tests applied a significance level of α = 0.05.
AcknowledgmentsWe would like to acknowledge D. Avisar and A. Kaplan for assistance and consultation for ICP-AES and B. Wozniak for ICP-MS; Y. Kauffmann for assistance with TEM/scanning TEM-EDS and FFT; E. Shimoni for cryopreservation of Entotheonella sp. and assistance with cryo-TEM sample preparation; and Z. Barkay for assistance with SEM-EDS. We acknowledge L. Roth, N. Kramer, and Ilan’s laboratory members for the support in laboratory and fieldwork. We are grateful to the Interuniversity Institute for Marine Sciences in Eilat (IUI) for ongoing support and use of their facilities. We acknowledge the European Synchrotron Radiation Facility for the provision of beam time on ID22. B.P. acknowledges the support of the Israel Discount Bank Academic Chair.
Funding: This work was supported by the Israel Science Foundation: grants 957/14 and 2157/22 (to M.I.).
Author contributions: Conceptualization: S.S., A.L., R.K., and M.I. Methodology: S.S., A.L., R.K., I.P., and M.I. Investigation: S.S., A.L., and R.K. Visualization: S.S., A.L., R.K., and I.P. Supervision: M.I. and B.P. Writing—original draft: S.S. Writing—review and editing: S.S., A.L., R.K., I.P., B.P., and M.I.
Competing interests: The authors declare that they have no competing interests.
Data and materials availability: Voucher sponge specimens are available at the Steinhardt Museum of Natural History (specimen number: Po26939-26942), Israel National Center for Biodiversity Studies at Tel Aviv University. All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials.
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