Microbiota of the alien species Paraleucilla magna (Porifera, Calcarea) from the Southwestern Atlantic, and a comparison with that of other calcareous sponges

Sponges (Porifera) co-evolved with microorganisms in a well-established symbiotic relationship. Based on this characteristic, sponges can be separated into high microbial abundance (HMA) and low microbial abundance (LMA) species. Paraleucilla magna (Calcarea, Porifera) is an alien species of ecological importance in the Brazilian coastline. Little is known about the composition of its microbiota and that of other calcareous species, especially those inhabiting the Southwest Atlantic. Here, we describe the microbiota of P. magna and compare it to that of other calcareous sponge species for which such data exist. P. magna’s microbiota shows a lower diversity than that of Clathrina clathrus, C. coriacea, Leucosolenia sp., Leuconia sp. and Leucetta antarctica. P. magna microbiota is dominated by two bacterial OTUs of the Alphaproteobacteria class, that could not be classified beyond class (OTU001) and family levels (OTU002; Rhodospirillaceae). The Thaumarcheota was the predominant archaeal phylum in P. magna, with OTUs mainly affiliated to the genus candidatus Nitrosopumilus. The comparison with other calcareous species showed that microbiota composition correlated well with sponge phylogenetic affiliation. Metabolic prediction with PICRUSt software of P. magna bacterial microbiota indicated that membrane transport and carbohydrate, amino acid and energy metabolisms were most abundant while, for the archaeal domain, pathways related to translation, and energy metabolisms were predominant. Predicted metabolic features were compared between the different sponge species and seawater samples, showing that pathways related to cell motility, membrane transport, genetic information processing, xenobiotics metabolism and signal transduction are higher in the former while amino acid and nucleotide metabolism, translation, replication and repair, folding, sorting and degradation and glycan biosynthesis and metabolism are abundant in the latter. This study shows that P. magna’s microbiota is typical of an LMA sponge and that it differs from the microbiota of other calcareous sponges both in its composition and in predicted metabolic pathways.

Atlantic. To date, few studies have analyzed the microbiota of calcareous sponges using 105 high-throughput technology and the only species for which such data exist are Clathrina 106 clathrus, C. coriacea, Leucosolenia sp., Leuconia sp. and Leucetta antarctica [7,8]. 107 In the present study, we aim to shorten this gap by describing the microbial     For microbiota analysis of the surrounding seawater, 5 liters of surface seawater were 142 collected in a sterile container and filtered through a 0.22 µm membrane (Millipore).

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The membrane was stored at -20 °C until processing.    Processing of the sequence reads and microbiota analysis 183 The sequence reads were processed with mothur software (v 1. 39      The parameters used for these analyses on the Calypso platform were the following: Data was filtered by removing rare taxa with less than 1% relative abundance, and the 261 data were normalized by total sum normalization followed by square root  of the total in seawater (Fig 2). Gammaproteobacteria isolated from a microbial mat found in a shallow submarine hot 341 spring off the coast of Japan [34], was also observed only in P. magna 6.

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OTUs found in the seawater sample were affiliated with a great diversity of 343 genera, and 35% of them did not reach a minimum relative abundance of 5%. Typical 344 marine genera were also observed such as those affiliated with the SAR and NOR 345 clades, with SAR_92 clade being the most abundant, with 14% of the OTUs (Fig 2).  (Fig 4).  Seawater samples showed greater microbiota diversity (Fig 7, Fig S3) than 411 sponges, and the sample from Fields Bay, Antarctica, was the least diverse among them.

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Among the sponge species, P. magna presented the lowest Shannon diversity index, 413 while all other samples presented higher and similar values (Fig 7). hyper saline mat (Fig 3A).

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The family Rhodospirillaceae, to which OTU002 was assigned, has 34 known 470 genera of Gram-negative microorganisms that present various nutritional strategies [41].

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In the phylogenetic tree constructed with related sequences retrieved from GenBank,

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OTU002 showed similarity with marine bacteria from different habitats, grouping 473 closely with a sequence from a bacterium isolated from a marine biofilm. 474 Yarza et al. [42] reported a phenomenon in which, as sequencing and data classification. This seems to be the case for OTU002. Differently, OTU001 appears to 478 have not been sequenced yet, as the highest similarity value with other reported 479 sequences in Genbank was 91%.

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The seawater sample showed the greatest microbial diversity, and 35% of the 481 OTUs did not reach a relative abundance of at least 5%. The most abundant OTU 482 belongs to the SAR92 clade, a member of the Gammaproteobacteria (Fig 2). This group in free-living bacteria. In our heatmap (Fig 5), we also observed a low abundance in 500 motility pathways.

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In the domain Archaea, the phylum Thaumarcheota was predominant in P. 502 magna samples, as already described by studies with other sponges (Fig 4)  subclass Calcaronea. We also analyzed the microbiota from four different seawater 538 samples to compare the microbiota of the sponges and the environment they inhabit, 539 which is fundamental for LMA sponge studies.

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The microbiota of all sponge species was clearly separated from those of 541 seawater samples both on the NMDS plot (Fig 6) and in the dendogram (Fig S2). together in the NMDS plot (Fig 6) and on the cladogram (Fig S2). These two sponges 547 species are of the same subclass as L. antarctica, however, no similarity in microbiota 548 composition was found, with the latter forming a separate branch on the cladogram. The 549 species Leucosolenia sp. and P. magna are of the same order, Leucosolenida, but they 550 did not cluster together. In fact, Leucosolenia sp., a species of the subclass Calcaronea, 551 clustered with species of Clathrina, i.e. species of the subclass Calcinea ( Fig S2).

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Although they are phylogenetically distant, they have the same type of aquiferous 553 system, asconoid. Paraleucilla and Leuconia samples also formed a cluster ( Fig S2) and 554 are in proximity on the NMDS graph (Fig 6). Both species have a leuconoid aquiferous 555 system.

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Although the microbiota of only a few calcareous sponges has been studied, the 557 clustering of asconoid and leuconoid species is very interesting. In the asconoid

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Hierarchical clustering based on the predicted bacterial metabolism showed that 574 sponges from the same genus share similar metabolic pathways and these are clearly 575 separate from seawater samples, indicating that there is a fundamental difference 576 between the microorganisms inhabiting these two habitats and the functions they 577 perform ( Fig S4). This can also be seen in the heatmap of the 15 most abundant 578 predicted pathways and in the statistical analysis with STAMP (Fig 8 and Fig 9,   579 respectively), in which functions related to nutrient acquisition (membrane transport, 580 xenobiotics biodegradation and metabolism) and symbiont/host interactions (cell 581 motility, genetic information processing and signal transduction) were prevalent in 582 sponges and functions related to cellular metabolism (amino acid metabolism) and 583 proliferation (nucleotide metabolism, replication and repair) had higher abundance in 584 seawater (Fig 9)