Enrichment of intracellular sulphur cycle –associated bacteria in intertidal benthic foraminifera revealed by 16S and aprA gene analysis

Abstract

Benthic foraminifera are known to play an important role in marine carbon and nitrogen cycles. Here, we report an enrichment of sulphur cycle -associated bacteria inside intertidal benthic foraminifera ( Ammonia sp. (T6), Haynesina sp. (S16) and Elphidium sp. (S5)), using a metabarcoding approach targeting the 16S rRNA and aprA -genes. The most abundant intracellular bacterial groups included the genus Sulfurovum and the order Desulfobacterales. The bacterial 16S OTUs are likely to originate from the sediment bacterial communities, as the taxa found inside the foraminifera were also present in the sediment. The fact that 16S rRNA and aprA –gene derived intracellular bacterial OTUs were species-specific and significantly different from the ambient sediment community implies that bacterivory is an unlikely scenario, as benthic foraminifera are known to digest bacteria only randomly. Furthermore, these foraminiferal species are known to prefer other food sources than bacteria. The detection of sulphur-cycle related bacterial genes in this study suggests a putative role for these bacteria in the metabolism of the foraminiferal host. Future investigation into environmental conditions under which transcription of S-cycle genes are activated would enable assessment of their role and the potential foraminiferal/endobiont contribution to the sulphur-cycle.

16S rRNA Gene Metabarcoding Indicates Species-Characteristic Microbiomes in Deep-Sea Benthic Foraminifera

“…The samples were stored in RNAlater solution in +8°C until DNA extraction (see Bird et al, 2017). Before extraction, the naked foraminiferal cells were again washed three times with sterile ASW to obviate any remaining shell material or residual RNAlater solution from hampering the downstream analysis (Bird et al, 2017;Salonen et al, 2019). By dissolving the shell and performing repeated washing prior and after that, we ensured that the extracted bacterial community was indeed intracellular and not shell associated.…”

“…Sediment DNA from the three replicates was extracted using the DNeasy PowerSoil ® DNA Isolation Kit (Qiagen, Germany). The DNA from foraminifera and sediment was amplified using universal bacterial primers 27F (5ꞌAGAGTTTGATCMTGGCTCAG) and 519R (5ꞌGTATTACCG CGGCTGCTG) targeting the variable regions V1-V3 of the 16S rRNA gene, in a similar way as described in Salonen et al (2019). Duplicate PCR reactions were performed and pooled in equal volumes, in order to ensure adequate amplicon volume for Illumina MiSeq library preparations and minimize PCR amplification bias.…”

“…A yet unresolved link in deep-sea foraminiferal ecology and environmental adaptation is the composition and role of their associated microbiomes. Benthic foraminifera are known to develop endobiotic relationships and close associations with prokaryotes (e.g., Bernhard, 2003;Bernhard et al, 2006, which may include denitrifiers (Bernhard et al, 2011), sulphur cycle-related bacteria (Tsuchiya et al, 2015;Salonen et al, 2019), Cyanobacteria (Prazeres et al, 2017), or even methanotrophs (Bernhard and Panieri, 2018). The endobiotic relationships between foraminifera and bacteria have been hypothesized to provide the host with metabolic flexibility, alternative carbon assimilation pathways, and enhanced nutrient/ organic compound cycling, enabling them to not only survive but also flourish in the harsh benthic environment (Bernhard et al, 2011;Bird et al, 2017;Prazeres et al, 2017;Tsuchiya et al, 2018;Salonen et al, 2019).…”

“…Despite the ecological importance of microbiome in foraminiferal adaptation, very little is known of its prevalence, significance, and evolution. Some studies indicate that the prokaryotic composition of the microbiome may be flexible and display a considerable amount of local variation (Prazeres et al, 2017) whereas evidence of speciesspecific microbiomes have also been observed in shallow intertidal areas with no indication of spatial variability (Salonen et al, 2019).…”

“…In the case of foraminifera, previous studies on their intracellular bacterial communities have been mainly based on transmission electron microscopy (TEM) observations (e.g., Koho et al, 2018). However, the current molecular ecology tools, such as DNA metabarcoding, offer new, efficient ways to investigate the diversity and composition of microbiomes of the unicellular foraminifera (e.g., Bird et al, 2017;Prazeres et al, 2017;Salonen et al, 2019).…”