Modeling and testing the physiology of DSi consumption

Live sponges were collected using the remotely operated vehicle (ROV) Remotely Operated Platform for Ocean Sciences (ROPOS) and taken to the laboratory for incubation in progressively increasing DSi concentrations (12, 30, 60, 100, 150, 200, and 250 M DSi; see Materials and Methods). Initially, all 11 assayed individuals increased their DSi consumption rate in response to the progressive increase of DSi availability in the seawater (Fig. 2, A and B, and tables S1 and S2). As also known for demosponges, the DSi consumption rate notably varied among individuals, with an average maximum consumption of 0.106 0.050 mol Si per milliliter of sponge tissue and per hour (hereafter given as mol Si ml1 hour1) at an average DSi concentration of 150.9 69.3 M. Over that concentration threshold, the consumption rate of most individuals did not increase with increasing DSi availability, revealing that the Si transport system reaches the maximum speed (i.e., optimal utilization) at about 150 M DSi and saturates at higher concentrations.

(A) DSi consumptions of 11 individuals of V. pourtalesii as a function of experimental silicic acid (DSi) concentration in the laboratory. The averaged response fits an ERM model (blue lines) better than a Michaelis-Menten (MM) kinetics (red lines). (B) Statistics of the average consumption (SD) best fitting to an ERM model. Note that DSi consumption calculated both from in situ incubations and from BSi produced under field conditions fall within the 95% confidence band of the model.

Unlike in all demosponges studied to date, the model best fitting the average DSi consumption rate of V. pourtalesii in response to DSi availability did not follow Michaelis-Menten kinetics (r2 = 0.841, P = 0.004; Fig. 2A). An exponential rise to a maximum (ERM) model showed the best fit (r2 = 0.898, P = 0.002; Fig. 2, A and B) to the empirical data on DSi consumption as a function of DSi availability, consumption rate = a (1 b[DSi]). Although the difference in statistical fit between the Michaelis-Menten and ERM models was apparently small, the ERM model was built on parameters with higher statistical significance (a = 0.093 0.008, P < 0.001; b = 0.978 0.006, P < 0.001) than those of the Michaelis-Menten model [Vmax = 0.114 0.019 mol Si ml1 hour1, P = 0.002; Michaelis constant (Km) = 44.876 23.934 M Si, P = 0.120]. The a parameter of the ERM model is the exact conceptual equivalent of the Vmax in the Michaelis-Menten model, indicating a maximum velocity of DSi utilization of 0.093 0.008 mol Si ml1 hour1. Likewise, the exact ERM equivalent of the Km parameter (i.e., the DSi concentration at which half-saturation or half Vmax is achieved) can also be calculated as [DSi] = log 0.5/log b, after having replaced in the ERM equation consumption rate = 0.5a. It yields a value of 31.16 M, revealing comparatively low affinity for the DSi in this sponge species (see Fig. 3).

(A) The kinetics of V. pourtalesii is compared against all other sponge species investigated to date (13, 15, 16, 18), which were demosponges with Michaelis-Menten kinetics. For relevant physiological comparison, DSi consumption rates were normalized to ash-free dry weight (AFDW), which represents essentially the organic component of the sponge that could be involved in silicification. The DSi consumption kinetics of V. pourtalesii, which does not follow a Michaelis-Menten model, is among the less efficient, except for that characterizing a group of slow-growing species in the genus Axinella. (B) A zoom on the graph within the range of natural DSi concentrations illustrates how V. pourtalesii is also less efficient than most demosponges at low DSi availability.

We tested the predictions of the developed kinetic model against empirical determinations of DSi consumption and BSi production rates in field conditions. Using five custom-manufactured methyl methacrylate chambers incorporating two ROV-operated seawater collectors, we incubated four sponge individuals and a control chamber under natural settings (Fig. 1, B to D, and movies S1 and S2). Incubations were conducted in the densest sponge aggregations of both the Sambro Bank Sponge Conservation Area and LaHave Basin (fig. S1) at depths of ~160 to 185 m, respectively, for periods varying from 19 to 28 hours, and at an average DSi concentration of 15.56 0.68 M. Individuals of different sizes were assayed (64, 126, 323, and 492 ml in volume; table S3), so that a relatively wide range of the size spectrum in the natural population was considered (all but very small or very large sponges). In situ consumption rates ranged from 0.007 to 0.034 mol Si ml1 hour1, averaging 0.024 0.012 mol Si ml1 hour1. This average consumption was markedly similar to the one predicted (0.027 0.006 mol Si ml1 hour1) by the laboratory kinetics at a DSi concentration of 15.56 M, falling within the 95% confidence range of the model (Fig. 2B).

We also estimated the rate at which BSithat is, the siliceous skeletonwas produced by the sponges under natural conditions to compare BSi production rates to DSi consumption rates obtained both from the in situ incubations and the predictions of the laboratory kinetic model. The recovery of two moorings that were immersed for 15 and 58 months brought up sponges that had settled on them, making the approach possible (Fig. 1, G and H; Materials and Methods). The two largest sponges on the mooring deployed for 15 months were about 14 months old, 1.4 and 2.9 cm in height, 1 and 3 ml in body volume, and 0.03 and 0.11 g in BSi content, respectively. The three largest sponges on the mooring deployed for 58 months were about 54 months old and ranged from 10 to 13 cm in height, 100 to 158 ml in volume, and 3.5 to 8.3 g in BSi content. These data indicated that skeletal BSi was produced at a rate threefold higher (0.056 0.008 mol Si ml1 hour1) during the first 14 months of life than in subsequent years (0.019 0.004 mol Si ml1 hour1), a growth pattern also known from other aquatic invertebrates (26). When the data from all five individuals were pooled together, an average BSi production rate of 0.033 0.021 M Si ml1 hour1 emerged. Again, this value fell within the 95% confidence range of the model prediction (0.029 0.007 M Si ml1 hour1) at a DSi availability of 16.93 M (Fig. 2B), which is the average DSi concentration in the bottom water on the central Scotian Shelf (table S4), as measured during a 20-year monitoring program (27). The general agreement among the rates of Si utilization predicted by the kinetic model, those measured through in situ incubations and those derived from the BSi production in field conditions, indicates consistency in the responses of the sponges in different situations, confirming that laboratory experiments are a suitable proxy for DSi utilization. It also suggests that, approximately, all consumed DSi (at least at natural ambient concentrations) becomes BSi production.

The DSi consumption kinetics of V. pourtalesii reaching optimal utilization at about 150 M DSi suggests that the natural population suffers from chronic DSi limitation, as mean DSi availability in the bottom water of the central Scotian Shelf averages only 16.93 8.65 M (table S4). Concentrations larger than 50 M have not been measured at any depth in the North Atlantic (17). A comparison of the DSi utilization kinetics known for sponges to date (Fig. 3) indicates that V. pourtalesii is comparatively less efficient in DSi consumption than all demosponges but Axinella spp. This suggests that to build and maintain the highly silicified skeletons characterizing hexactinellid sponges, they may need continuous exposure to DSi levels higher than those characterizing the modern photic ocean [<10 M; (1)]. The question remains, however, as to why the DSi consumption system of these sponges persists largely maladapted, unable to evolve in response to a shrinking DSi availability that started in the oceans at least 60 million years (Ma) ago (19, 28) or even earlier (29).

To elucidate why hexactinellid sponges are particularly inefficient when using DSi at the relatively modest concentrations of the modern ocean, we attempted to activate and identify in V. pourtalesii (see Materials and Methods) the Si transporters potentially involved in the process of both DSi uptake and its internal transport. To this end, we quantified gene expression in six of the sponge individuals that had been exposed to progressive DSi enrichment (from 12 to 250 M DSi) during the kinetic experiment, as indicated in the Ex situ incubations for kinetics of DSi consumption section. Their gene expression was contrasted with that of six individuals not exposed to any DSi enrichment but to the natural (12 to 17 M DSi) concentration (hereafter referred to as control individuals). The set of treated individuals (hereafter referred to as DSi-enriched individuals) consisted of the sponges #3, #4, #5, #7, #9, and #10 used in the kinetic experiment (as indicated in Fig. 2A).

A de novo reference transcriptome was obtained after pooling the reads from the 12 sponge libraries, and its resulting summary metrics (table S5) showed it to be well assembled and with very high BUSCO completeness scores (95.1% of the eukaryotic cassette and 87.3% of the metazoan cassette). In the DSi-enriched individuals, 597 genes were differentially up-regulated relative to the control group, of which 269 had a BLAST hit against the RefSeq and 197 against Swiss-Prot databases (fig. S2 and data file S1). In the control, 980 genes were found up-regulated when compared with the DSi-enriched individuals (fig. S2 and data file S1). Among the genes up-regulated in the DSi-enriched individuals, only 131 (33%) had a gene ontology (GO) term annotation. Identified overexpressed genes in the DSi-enriched individuals belonged to a wide array of functional categories (fig. S3 and table S5). We identified abundant transmembrane transport and vesicle-mediated transport categories, as well as responses to stress, lipid metabolism, and mRNA processing, among others in the Biological Process category. In addition, certain molecular functions were up-regulated, such as lysosome-related genes (e.g., solute carriers and cathepsins among others), transporter activity, chitin binding, and oxidoreductase activity (Fig. 4A and fig. S3). Indirect evidence indicates that silicification in sponges is a complex, energy-consuming biological process (10, 12). Therefore, up-regulation of multiple gene pathways not directly related to Si utilization was not unexpected. For our study, we focused only on those genes that had previously been demonstrated in the preexisting literature of other organisms as being involved either in Si transport or in Si polymerization. This circumvents the need to carry out further unrealistic heterologous expressions or knockout experiments with V. pourtalesii, a deep-sea animal for which any subsequent gene functionalization would require additional unaffordable economic and logistic transnational investments for additional experimental work with live individuals.

(A) Heatmap of the genes putatively related to silicon (Si) utilization and ion transporters. Relative expression was obtained from normalized expression levels using trimmed mean of M-values (TMMs) of potential target genes in biomineralization. Genes DE are shown in red bold letters. (B) Normalized expression levels using TMMs of genes known to be involved in Si processing in sponges or other organisms. Asterisks indicate DE genes with statistical significance between the two groups of individuals (DSi-enriched group versus control group) following the criterion of at least twofold expression and a P value corrected by false discovery rate (FDR) of 0.001. SD, standard deviation.

We found two homologs of the gene glassin, which code for the only silicifying protein identified in hexactinellids to date (22). Unexpectedly, only glassin 1 was slightly up-regulated (see Discussion) but not differentially expressed (DE), with its expression level in the DSi-enriched group being barely twofold that of the control group (Fig. 4). Silicatein genes, which are members of the cathepsin family of cysteine proteases and code for the silicifying enzyme of demosponges, had occasionally been reported from hexactinellids (30, 31). However, no silicatein sequences were found in V. pourtalesii, a result consistent with the growing suspicion that initial reports of silicatein in hexactinellids were either contamination from demosponge samples or misidentified cathepsin-like proteins not involved in the silicification (22, 32).

Two gene groups of transmembrane proteins related to Si transport (aquaporins and ArsB) were up-regulated in the DSi-enriched individuals. Aquaporins are ancient channel proteins that facilitate bidirectional passivebut relatively selective (33)flux of water and/or small noncharged solutes across membranes and that are present in all kingdoms of life (34). The overexpressed aquaporins consisted of three genes (Figs. 4 and 5A). One of the V. pourtalesii protein sequencesaquaporin 3likeshowed high similarity to aquaglyceroporin 3 of chordates, a second oneaquaporin 9likewas more similar to the aquaglyceroporin 9 of chordates, while a third oneaquaporin 3/9likehad equal sequence similarity to both aquaglyceroporin 3 and 9. Only this later aquaglyceroporin 3/9 showed a much higherand statistically significantoverexpression in the DSi-enriched individuals (Fig. 4). Aquaglyceroporins 3 and 9 are known to be major intrinsic proteins that function in chordates as passive channels facilitating a Si inflow from the intercellular medium into cells (35).

(A) Phylogenetic hypothesis of aquaporin protein family relationships and (B) low-silicon (Lsi2) and arsenite-antimonite (arsB) efflux transporters, which are also related to the protein family pink-eyed dilution (PED) transporters. In both cases, phylogenetic trees were obtained with ML, and the topology was congruent with that obtained from a Bayesian inference analysis. Therefore, posterior probabilities from the Bayesian inference were mapped on the nodes. Only bootstrap values more than 70 and posterior probabilities more than 0.90 are shown on the nodes. Accession numbers and contig names are in parentheses. Names in blue are new data from this study.

In a maximum likelihood (ML), noncomprehensive, phylogenetic analysis of the aquaporin protein family, which produced a tree topology with 100% congruence to that of an alternative Bayesian approach, four main clades were obtained (Fig. 5A and fig. S4): (i) one containing the aquaglyceroporins 3, 7, and 9 as well as the plant nodulin26 transporters (NOD26) and Lsi1 sequences from plants, which are all involved in passive transport of silicic acid, glycerol, and possibly water to the cells; (ii) a clade containing plasma membrane intrinsic (PIP) and tonoplast intrinsic (TIP) aquaporins, involved in water, glycerol, and ammonia transport; (iii) a clade containing aquaporin 8 (present in some sponges but not in hexactinellids) and small basic intrinsic proteins (SIP), mediating water, ammonia, and urea passive flow; and (iv) a clade of several aquaporins and PIPs involved in water transport (Fig. 5A). Only the first clade, recovering aquaglyceroporins 3, 7, and 9, showed robust nodal support.

The three V. pourtalesii aquaglyceroporin sequences involved in DSi transport also clustered (with statistical significance) with other unnamed aquaporins that we have recovered from the transcriptome of the hexactinellid Rosella fibulata and several demosponges, including both silicifying (i.e., Lubomirskia baikalensis, Cliona varians, and Petrosia ficiformis) and nonsilicifying species (i.e., Dendrilla antarctica and Ircinia fasciculata; see Discussion), collectively forming a robust sponge clade (Fig. 5A and fig. S1). While demosponges had a single member of this aquaporin clade (3, 7, and 9), hexactinellids showed expansion into three members (Fig. 5A), which could be the result of gene duplication and initial subfunctionalization for improved Si transport (see Discussion).

The other gene group related to Si utilization that was differentially overexpressed in the DSi-enriched individuals were arsB- and/or Lsi2-like genes (Figs. 4 and 5B), members of a superfamily of active ion transporters (Na+/H+ antiporters) that mediate a selective efflux of metalloids from the cytoplasm. The independent discovery of the arsB genesinitially described from bacteria as for transmembrane transporters of arsenic (36) and other metalloids (37)and the Lsi2 (low silicon 2) genes (38, 39)first identified in plants, as coding for transmembrane transport of Si and other metalloidsfavored two different gene denominations (i.e., arsB versus Lsi2). This historical nomenclature still persists despite these genes showing clear sequence orthology and strong similarity in function (see Discussion). Two arsB/Lsi2 genes (herein referred to as arsB 1 and arsB 2) were found differentially overexpressed in V. pourtalesii (Fig. 4). ArsB genes also occur in the demosponge Amphimedon queenslandica and are known in several silicifying eukaryotes (Fig. 5B and fig. S4), including diatoms, radiolarians, and choanoflagellates (23). These eukaryotic versions of Lsi2 and arsB genes also show sequence similarity to the prokaryotic arsenic transporters (Fig. 5B). In our phylogenetic hypothesis for the evolution of arsB/Lsi2 transporter proteins, in which the ML and the Bayesian approaches showed complete tree topology congruence (Fig. 5B), four main subclades were recognized among the arsB/Lsi2 proteins: (i) one consisting of the arsB homologs of bacteria, with a single arsB domain; (ii) another large clade composed by the homologs of diatoms and plants, with the diatom sequence containing a CitMHS domain [citrate-Mg2+:H+(CitM)-citrate-Ca2+:H (CitH) symporter] and the plant homologs both Nhab and arsB domains; (iii) a small clade containing the Lsi2-like homologs of choanoflagellates, which only have an arsB domain; and (iv) a large clade containing the animal homologs of arsB/Lsi2, with a single CitMHS and one or several transmembrane domains.

Of note, SITs, which are sodium-coupled transmembrane proteins operating as active silicic acidspecific transporters in a variety of unicellular silicifying eukaryotes (23), such as diatoms, choanoflagellates, and haptophytes, were absent in V. pourtalesii, in agreement with other studies on sponge genomes (23). Related silicon transporterlike genes (SIT-Ls), occurring in some metazoans such as annelids, copepods, and tunicates (23), were also absent. Likewise, the singular active Si transporter of vertebrates, solute carrier transporter Slc34a2 (40), was also absent. The NBC (Na+/HCO3) transporter, which was tentatively suggested to be involved in cotransporting DSi in the demosponge Suberites domuncula (24), was present in the transcriptome of V. pourtalesii (TRINITY_DN38500_c0_g1_i1) but not up-regulated by DSi enrichment, a response that does not support a direct involvement in DSi transport in this hexactinellid sponge.

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Cooperation between passive and active silicon transporters clarifies the ecophysiology and evolution of biosilicification in sponges - Science...