Sample Processing Impacts the Viability and Cultivability of the Sponge Microbiome

Abstract

Sponges host complex microbial communities of recognized ecological and biotechnological importance. Extensive cultivation efforts have been made to isolate sponge bacteria, but most still elude cultivation. To identify the bottlenecks of sponge bacterial cultivation, we combined high-throughput 16S rRNA gene sequencing with a variety of cultivation media and incubation conditions. We aimed to determine the extent to which sample processing and cultivation conditions can impact bacterial viability and recovery in culture. We isolated 325 sponge bacteria from six specimens of Cymbastela concentrica and three specimens of Scopalina sp. These isolates were distributed over 37 different genera and 47 operational taxonomic units (defined at 97% 16S rRNA gene sequence identity). The cultivable bacterial community was highly specific to its sponge host and different media compositions yielded distinct microbial isolates. Around 97% of the isolates could be detected in the original sponge and represented a large but highly variable proportion (0.5–92% total abundance, depending on sponge species) of viable bacteria obtained after sample processing, as determined by propidium monoazide selective DNA modification of compromised cells. Our results show that the most abundant viable bacteria are also the most predominant groups found in cultivation, reflecting, to some extent, the relative abundances of the viable bacterial community, rather than the overall community estimated by direct molecular approaches. Cultivation is therefore shaped not only by the growth conditions provided, but also by the different cell viabilities of the bacteria that constitute the cultivation inoculum. These observations highlight the need to perform experiments to assess each method of sample processing for its accurate representation of the actual in situ bacterial community and its yield of viable cells.

Integrated metabolism in sponge–microbe symbiosis revealed by genome-centered metatranscriptomics

“…The 16S rRNA sequence from CcThau was 99% similar to the sequence from Candidatus Nitrosopumilus sediminis AR2 (GenBank: CP003843.1). According to studies based on OTUs recovered from metagenomic data sets and 16S rRNA gene sequencing, these three organisms are among the most dominant members of the C. concentrica microbiome (Thomas et al, 2010;Fan et al, 2012;Esteves et al, 2016). For example, based on 16S rRNA gene sequencing, the Phyllobacteriaceae OTU may account approximately 17% of all bacteria in the sponge, while the Nitrospira OTU has a relative abundance of~6% (Thomas et al, 2010).…”

“…Such nutrients are likely found across the sponge tissue, which is consistent with a scattered distribution of CcPhy cells throughout the mesohyl (see Figure 7; Supplementary Figure S10). Considering the high relative abundance of Phyllobacteriaceae within the symbiont community Esteves et al, 2016), CcPhy may thus be a major contributor to uptake and utilization of DOM by the microbiome. The DOM used by CcPhy may come from the sponge's filtering activity (Figure 1a;de Goeij et al, 2008de Goeij et al, , 2013 or from diatoms ( Figure 1b) that were found throughout the mesohyl (Figure 7), especially considering that axenic cultures of diatoms have been shown to release between 5% and 21% of their total fixed carbon as dissolved organic carbon (Wetz and Wheeler, 2007).…”

“…The sponge Cymbastela concentrica contains consistently dominant and uncharacterized alphaproteobacterial symbionts, particularly within the family Phyllobacteriaceae (Taylor et al, 2004;Fan et al, 2012;Esteves et al, 2016) as well as community members belonging to the Gammaproteobacteria, the Nitrospira and the archaeal phylum Thaumarchaeota among others Esteves et al, 2016). In addition, C. concentrica hosts an abundant population of diatoms (Taylor, 2005).…”

Allelochemicals Produced by Brown Macroalgae of the Lobophora Genus Are Active against Coral Larvae and Associated Bacteria, Supporting Pathogenic Shifts to Vibrio Dominance

“…Each branch was first soaked in calcium-and magnesium-free seawater (CMFSW; NaCl [450 nM], KCl [10 mM], Na 2 SO 4 [7 nM], NaHCO 3 [0.5 mM]) for approximately 1 h to aid in the removal of tissue from the skeleton (88). The branch was then sprayed with pressurized air from a blow-gun attached to a SCUBA cylinder and fitted with a custom sterile tip (89).…”

Natural Products from Marine Invertebrates and Microorganisms in Brazil between 2004 and 2017: Still the Challenges, More Rewards

“…26 Such evidences are supported by solid exprimental data indicating both vertically and horizontally bacterial transmission within and between sponge species. 21,22,26,31 The importance of developing strategies for culturing marine bacteria, in particular sponge symbionts, must be stressed, 21,22,26,32 since the large majority of sponge-associated microbial strains are considered to be resistant to cultivation 26 or the secondary metabolism is difficult to be trigger. This is not only particularly relevant for the correct assignment of a microbial production of secondary metabolites, 30 along to the knowledge of microbial physiology and ecology of sponge symbionts 22,26,31 but also for the detailed investigation of unique biosynthetic pathways which are not found elsewhere in nature but in sponges.…”