Co-cultivation of the marine sponge Halichondria panicea and its associated microorganisms

Stephen Knobloch,Ragnar Jóhannsson,Viggó Marteinsson

doi:10.1038/s41598-019-46904-3

Abstract

Marine sponges host bacterial symbionts with biotechnological potential, yet isolation of true sponge symbionts remains difficult due to their host dependency. Moreover, attempts to grow sponges for their pharmacologically-active compounds outside of their habitat often results in a shift of their microbial community. In this study we evaluate suitable sponge cultivation methods that allow maintenance of both the marine sponge Halichondria panicea and its associated bacteria in an ex situ environment. In addition, we present a method for co-cultivation of sponge explants and microbes separated by a membrane in a multi-chamber device. Tests on ex situ cultivation of H. panicea under different controlled conditions showed that only high water exchange rates in the aquarium enabled maintenance of its dominant symbiont “Candidatus Halichondribacter symbioticus” at a high relative abundance in the sponge body, a prerequisite for co-cultivation. The bacterial enrichment retrieved from co-cultivation contained bacteria from nine different classes in addition to sequences corresponding to “Ca. H. symbioticus”. This represents an increase of the cultivable bacterial classes from H. panicea compared to standard isolation techniques on solid media plates. The current study provides insights into sponge-microbe maintenance under ex situ conditions and proposes a new method for the isolation of sponge-associated bacteria.

Highlights

Marine sponge have been in the spotlight of marine derived natural product discovery for the past decades due to their rich inventory of pharmacologically-active compounds[1,2,3]
H. panicea from various locations in the North Atlantic host the dominant bacterial symbiont “Candidatus Halichondribacter symbioticus” which often constitutes over half of the relative bacterial abundance in the sponge body[27,28,29,30,31]
In this study we evaluate suitable sponge cultivation methods which allow maintenance of both the sponge and its associated bacteria in an ex situ environment, and present a new sponge-bacteria co-cultivation method for isolation of sponge-associated bacteria which can be used under laboratory setting

Summary

Introduction

Marine sponge (phylum Porifera) have been in the spotlight of marine derived natural product discovery for the past decades due to their rich inventory of pharmacologically-active compounds[1,2,3]. Other bacteria within the sponge body vary depending on geographical location and time of sampling[27,29] Despite variability they exhibit a clear difference in diversity from the surrounding seawater bacterial community, showing that, apart from “Ca. H. symbioticus”, H. panicea hosts other sponge-associated bacteria[28]. The chambers are inoculated with single microbial cells or microbial suspensions and are deployed back into their natural environment, providing the complex environmental conditions needed for growth[39] Such systems have been, most notably, applied for the isolation of soil microorganisms[37,40], but have been implanted in wild sponges, yielding novel so far uncultivated microorganisms[41]. We apply 16S rRNA gene amplicon sequencing along with standard plating techniques to analyse the bacterial diversity in H. panicea during cultivation and compare the enriched bacterial fraction from co-cultivation to the results from standard plating

Methods

Results

Discussion

Conclusion