Hologenomic insight into the marine sponge Haliclona fulva.

Among symbiotic relationships, there are benefit associations between organisms sharing the same space. This relationship has been detected in animals at different taxonomical levels and evolutionary history, and it has originated the concept of holobiont, which is defined as the host and its symbionts. Symbionts can be constant or inconstant, can be vertically or horizontally transmitted, and they can play a harmful, harmless, or helpful functional role [1. The most archaic examples of holobiont, is the one formed by marine sponges (Phylum Porifera). They represent an ancient and primitive lineage of metazoans, with earliest fossil records detected in Precambrian formations [2. These multicellular organisms had been inhabiting Earth for at least 590 million years, and are adapted to thrive in a diverse range of aquatic environments (from shallow coastal waters to very deep zones to freshwater) [3 and are found across different latitudes varying from tropical to artic regions [4, 5. This complex symbiosis in sponges is responsible to achieve functions as participation in biogeochemical fluxes, production of secondary metabolites, mechanisms providing protection against predators and epibionts, or the capacity to alter the water column, among others [6. Thus far, microbial diversity of many sponges have been well characterized for Eubacteria and Archaea, relying on the massive sequencing and analyses of 16S rRNA gene amplicons. From these surveys it is known that this bacterial symbiotic component includes more than 40 phyla, being the most dominant Proteobacteria, Chloroflexi, Thamumarchaeota, Poribacteria, Cyanobacteria, Actinobacteria among others [7, 8. However, while much progress has been made about diversity and patterns of sponge-associated microbial communities, information on the metabolic pathways, functional role on each counterpart and the genomic analysis as a whole is scarce. Use of omics-sciences have become one of the most powerful strategies to understand complex interaction between sponge and associated symbionts [9. Among them, High Throughput Sequencing-based metagenomics is considered a valuable and informative tool to attain an improved resolution on microbial community composition and expand the coverage of hologenome components that are otherwise overlooked when using a single gene marker amplicon sequencing (e.g.,16S rRNA gene) [10. Total metagenomic DNA sequencing has been reported to detect and characterize complex and unknown viral communities (viromes) [11, 12. In fact, this technique and the viral genomes derived have been already recognized by the International Committee for Taxonomy of Viruses [12 as a standard for listing and identifying viruses. Metagenomics also allows discovering genes and novel metabolic pathways [13. Despite of advantages provided by omics, only few sponges have been analyzed through metagenomic and transcriptomic approaches, some of them are: Amphimedon queenslandica [14 [15, Aplysina aerophoba [10, Cliona varians [16, Cymbastela concentrica [17, 18, Rhopaloides odorabile [18, 19, Stylissa carteri [20 and Xestospongia muta [21. On the Mediterranean Sea, the sponge Haliclona fulva (Demospongiae: Haplosclerida), is an abundant and thickly encrusting marine sponge species dwelling in shaded habitats such as coralligenous accretions or semi-dark caves, between 5 and 50 m depth [22. H. fulva is considered as a promissory species due to the nature of its metabolites (terpenoids, polyacetylenes, alkaloids, peptides and polyketides) [23, 24, some of them have the potential to be used as taxonomical markers and they have exhibited antitumoral and antimicrobial activities. At microbiological level, its symbiotic microbial community was recently described by analyzing 16S rRNA gene hypervariable region V4 amplicon. Results showed that it is particular and distinctive among species inside the same genus, and it is formed mainly by two symbionts belonging to the orders Nitrosomonadales and Cenarchaeales [25. To better understand the dynamic of symbionts, to uncover others overlooked members whose ecological role might be essential to maintain sponge¿s fitness, and to gain insight into metabolic functions coded by the holobiont, DNA of the holobiont was sequenced. This part was carried out in the framework of doctoral thesis of Erika Garcia-Bonilla at Universidad Nacional de Colombia. A preliminary analysis by using MG-RAST (metagenomic rapid annotations using subsystems technology) pipeline [26, interestingly showed that the microbial community was dominated by viruses (39.6%), followed by Bacteria (37.3%), Eukaryota (21.9%) and Archaea (0.51%). Remarkably, in the case of virus, the obtained sequences did not match against known representative sequences, which is considered to be normal due to the lack of a representative database of marine viral genomes [11. Those results indicate that the sponge has an untapped reservoir of new viruses. In relation to function, sequences matched mainly genes of metabolism. These genes are involved in pathways related to carbohydrates, amino acids, proteins, cofactors, vitamins and respiration. Other important observation was the presence of phages, prophages, transposable elements and plasmids, which is in agreement with the high percent of viruses found in the metagenome. As was mentioned above, preliminary results shown in this proposal are in an initial stage of data analysis, for that reason, the main objective of this project is to identify virome associated with Haliclona fulva. The characterization includes identification of virus, assembly of genomes, understand how virus could interplay with dominant symbionts and the sponge, and finally to state hypothesis about their ecological role. For that proposal, the project establishes a collaboration network among investigators of different research groups (UNESIS: Strategic Marine Ecosystems and Plant and crop biology groups both at Pontifical Xaverian University, Colombia; Microbiomas Foundation: Microbial Ecology, Metabolism, Genomics & Evolution - Diversity, Ecogenomics & Holobionts, Colombia; and Computational, Evolutionary and Systems Biology Group at University of Sao Paulo, Brazil), whose complementary knowledge and expertise will help to reach the objectives described above. So far, to our best knowledge, there is not information about H. fulva metagenome, so this project can contribute to decipher diversity and ecological role of microbiome, to identify novel viral genomes, and try to elucidate if there is any relationship between abundances of identified microbial phylotypes and viruses types. Understanding that dynamic can shed light about possible responses of marine sponges, its biodiversity and impact on biogeochemical cycling in a variable environment where life is under increasing threat (global warming, acidification, pollution). The execution of this project will provide new knowledge, training in cutting-edge technologies in functional metagenomics and bioinformatics and strengthening of research collaborations, which open opportunities to the future development of similar projects for tropical marine environments, a field that remains almost unexplored. Therefore, expanding our knowledge on H. fulva is of taxonomical, chemical, biotechnological and environmental interest.