Abstract
Bacteriophages play a fundamental role in microbiome function, yet their diversity in association with marine invertebrates remains largely unexplored. The microbiomes of these invertebrates are also poorly understood, despite the critical roles microbes play in host health and function, and the downstream consequences of these microbiomes for human health through ecosystem services. Bryozoans are sessile marine invertebrates found worldwide and act as important habitat-forming species, providing shelter for other marine organisms. Several bryozoan species are also recognized as invasive, including within New Zealand. This study presents the first isolation and characterization of phages from bryozoans, representing the first evidence of cultivable phages within the entire phylum.
Three bryozoan species, Bugulina flabellata, Tricellaria catalinesis, and Watersipora subatra were collected from multiple sites along Otago Harbour, Dunedin, New Zealand. Species identification was confirmed to the species level using scanning electron microscopy. Phages were isolated through direct plating with Escherichia coli K12 as the host, and morphology and structural features were determined using transmission electron microscopy. To further investigate the microbial community, draft metagenomic assemblies were generated for B. flabellata and W. subatra.
Six phages were isolated, hereafter referred to as Anaranjado, Beguni, Camembert, Halloumi, Fondue, and Petitebasque. Of these, Beguni and Anaranjado underwent sequencing, annotation, and mass spectrometry, enabling the identification of proteins of interest and comparisons with other E. coli phages. The 2.84 Å 3D atomic capsid map of phage Beguni was reconstructed using cryogenic electron microscopy.
Metagenomic analyses revealed high bacterial diversity in the bryozoan species B. flabellata and W. subatra, dominated by Proteobacteria and Bacteroidetes. Viral content waslimited, with Caudoviricetes as the primary viral family. Among the six isolated phages, four were siphophages and two were myophages. Genomic analyses confirmed their virulent, lytic nature, with lysis proteins present in both Anaranjado and Beguni. Major capsid protein (MCP) modeling showed similarity of Beguni MCP to the HK97 fold, while 3D reconstruction and modeling also identified putative decoration proteins. Together, genomic and structural analyses indicate that these bryozoan phages share similarities with other E. coli phages. These findings establish the first evidence of cultivable phages associated with bryozoans and provide new genomic and structural resources, while also highlighting bryozoans as a previously overlooked reservoir of viral diversity. Finally, this study lays the foundation for future research on phage-host interactions within the microbiomes of this phylum and other marine invertebrates.