An AOA-Dominant Microbiome in a Novel Deep-Sea Glass Sponge Species from the South China Sea: Symbiotic Diversity, Adaptation, and Network Interactions

Marine sponges, ancient members of benthic communities, harbor diverse prokaryotic communities crucial for their ecology. While shallow-water sponge microbiomes are extensively studied, deep-sea sponge microbiomes remain less understood. Deep-sea sponges play vital ecological roles, including microhabitat provision, nutrient cycling, and benthic-pelagic coupling. Previous research has highlighted the importance of ammonia-oxidizing archaea (AOA), nitrite-oxidizing bacteria (NOB), and sulfur-oxidizing bacteria (SOB) in deep-sea sponge nutrient conversions. Furthermore, the increasing attention on sponge-associated viromes underscores the importance of viruses in regulating the holobiont. This study focuses on a novel deep-sea glass sponge species from the South China Sea to explore its microbiome's genomic composition and interactions, particularly the potential roles of AOA, *Bdellovibrio*, and phages within this unique ecosystem. The goal is to gain a comprehensive understanding of the metabolic potential and mutualistic strategies of deep-sea sponge symbionts.

Extensive research has explored the diversity and function of shallow-water sponge microbiomes, revealing a complex interplay between the host and its associated microbes. Studies have identified a wide range of prokaryotic phyla, highlighting the functional importance of symbiotic relationships in nutrient cycling and other processes. Deep-sea sponges are recognized for their unique adaptations and ecological significance, yet their microbiomes are less characterized. Studies on specific deep-sea species, such as *Lophophysema eversa* and *Vazella pourtalesii*, have begun to reveal the metabolic capabilities of their associated microbes, including complete nutrient cycles. However, a comprehensive understanding of deep-sea sponge microbiomes across different species and environments remains limited. Recent research has also started to investigate the role of viruses in sponge holobionts, focusing primarily on shallow-water species and highlighting the importance of phage-bacteria-host interactions.

A deep-sea glass sponge (*Bathydorus* sp. SQW35) was collected from the South China Sea at 983 m depth during the R/V *Tansuoyihao* TS-7 cruise. The sponge was cleaned, and DNA was extracted from two tissue pieces. Metagenomic libraries were constructed and sequenced using Illumina HiSeq2500, generating 2 × 150 bp paired-end reads. Raw reads were processed using Fastp and FastUniq. 16S rRNA gene sequences were predicted and analyzed using rRNA_HMM and QIIME1 pipelines for microbial community analysis. Clean reads were assembled using SPAdes and MEGAHIT, followed by genome binning using MetaWRAP and dereplication using dRep. The quality of the resulting 14 MAGs (completeness >50%, contamination <10%) was assessed using CheckM, with taxonomic annotation and RED calculation performed using GTDBtk. Relative abundance of MAGs was estimated using coverM after removal of eukaryotic reads identified by EukRep. The global distribution of the dominant symbiont was investigated by BLASTN against the SMP and D-SMP databases. Genome annotation was performed using Prodigal, KofamScan, PfamScan, and BLASTP. Phylogenetic analyses were conducted using IQ-TREE with MAFFT alignment and trimAl trimming. Phage-like contigs were identified using several tools including DeepVirFinder, VIBRANT, VirFinder, VirSorter, VirSorter2, and CheckV, with taxonomic assignment performed using vConTACT2.

The *Bathydorus* sp. SQW35 sponge was identified as a potentially novel species based on its coxI gene. 16S rRNA gene analysis revealed a highly consistent prokaryotic community dominated by *Nitrososphaerota* (81.81 ± 2.09%), mainly belonging to the *Nitrosopumilaceae* family (AOA). Gammaproteobacteria (SOBs) were the second dominant group (10.60 ± 0.85%). Fourteen high-quality MAGs were recovered, thirteen representing potentially novel species. The dominant symbiont was AOA MAG B01 (70.31 ± 1.15% of metagenomic reads), closely related to the *Cenarchaeum* clade but representing a likely novel species and genus. Two *Bdellovibrionota* MAGs (B11 and B12) showed characteristics consistent with symbiotic evolution, including smaller genome size and lower GC content compared to free-living relatives. Analysis of carbon, nitrogen, and sulfur metabolisms revealed the presence of various pathways in the microbial consortium. Many ELPs, including Ank and TPR domains, were identified in several bacterial MAGs but not in AOA MAGs. The presence of CRISPR-Cas systems, particularly in the dominant symbionts, indicated their potential role in phage defense. Analysis revealed a diverse phage community within the sponge, with a high abundance of phage fragments.

The study's findings significantly advance our understanding of deep-sea sponge microbiomes. The AOA-dominant microbiome of *Bathydorus* sp. SQW35 is consistent with observations in other deep-sea sponges, highlighting the importance of AOAs in nutrient cycling. The discovery of a novel AOA species expands the diversity of sponge-associated AOAs, providing insights into their adaptation to the deep-sea environment. The identification of *Bdellovibrio* species as sponge symbionts and their apparent reductive genome evolution reveal new aspects of their symbiotic lifestyles. The prevalence of CRISPR-Cas systems and ELPs suggests their roles in defense against phages and host-symbiont interactions, respectively. The identification of a large phage community indicates a complex and dynamic interplay within the sponge holobiont. This study underscores the complexity and unique adaptations of deep-sea sponge microbiomes.

This study provides a comprehensive genomic analysis of the microbiome of a novel deep-sea glass sponge species, *Bathydorus* sp. SQW35, revealing an AOA-dominant community. The study identified novel species within the AOA and *Bdellovibrio* groups and demonstrated the roles of CRISPR-Cas systems and ELPs in adaptation. Future research should investigate the functional roles of the identified phages and the intricate interactions within this complex symbiotic network, expanding our understanding of deep-sea ecosystem functioning.

The study is based on a single sponge individual, limiting the generalization of findings. The absence of parallel analysis of seawater samples prevents definitive conclusions about the origin of the identified viruses and phages. Further research with more extensive sampling is necessary to fully elucidate the diversity and distribution of the sponge-associated microbiome and virome.