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Ascidians (Chordata, Ascidiacea) are considered to be prominent marine invaders, able to tolerate highly polluted environments and fluctuations in salinity and temperature. Here, we examined the seasonal and spatial dynamics of the microbial communities in the inner-tunic of two invasive ascidians, Styela plicata (Lesueur 1823) and Herdmania momus (Savigny 1816), in order to investigate the changes that occur in the microbiome of non-indigenous ascidians in different environments. Microbial communities were characterized using next-generation sequencing of partial (V4) 16S rRNA gene sequences. A clear differentiation between the ascidian-associated microbiome and bacterioplankton was observed, and two distinct sets of operational taxonomic units (OTUs), one core and the other dynamic, were recovered from both species. The relative abundance of the dynamic OTUs in H. momus was higher than in S. plicata, for which core OTU structure was maintained independently of location. Ten and seventeen core OTUs were identified in S. plicata and H. momus, respectively, including taxa with reported capabilities of carbon fixing, ammonia oxidization, denitrification, and heavy-metal processing. The ascidian-sourced dynamic OTUs clustered in response to site and season but significantly differed from the bacterioplankton community structure. These findings suggest that the associations between invasive ascidians and their symbionts may enhance host functionality while maintaining host adaptability to changing environmental conditions.

Various DNA extraction methods are often used interchangeably for the characterization of microbial communities despite indications that different techniques produce disparate results. The microbiomes of two ascidian species were herein characterized using two common DNA extraction kits, the DNeasy Blood and Tissue Kit (Qiagen) and the PowerSoil DNA Isolation Kit (Mo Bio Laboratories), followed by next-generation (Illumina) sequencing of partial 16S rRNA genes. Significant differences were detected in microbial community diversity and structure between ascidian species, but not between kits, suggesting similar recovery of biological variation and low technical variation between the two extraction methods for ascidian microbiome characterization.

Harbor systems represent passive gateways for the introduction of nonnative ascidians that compete with the surrounding benthos and may spread through localized dispersal, even populating adjacent natural reefs. To investigate the potential role of microbial symbionts in the success of ascidian introductions and spread, we evaluated the host-specificity of prokaryotic communities within two ascidian species commonly found off the North Carolina coast. Replicate samples of the native ascidian Eudistoma capsulatum, the nonnative ascidian Distaplia bermudensis and seawater were collected from artificial (harbor) and natural reef substrates. Prokaryotic communities in seawater samples and ascidian tunics were characterized via next-generation sequencing of partial 16S rRNA gene sequences. Ascidian microbiomes clustered strongly in response to host species, with significant differences in community structure between the two species and seawater. Further, symbiont community structure differed significantly between E. capsulatumindividuals collected from artificial and natural habitats, though this was not the case for D. bermudensis. These findings suggested that some ascidian species possess stable microbial symbiont communities that allow them to thrive in a wide range of habitats, while other species rely on the restructuring of their microbial communities with specific symbionts (e.g. Chelativorans) to survive under particular environmental conditions such as increased pollution., (© FEMS 2018.)

Many ascidian species have experienced worldwide introductions, exhibiting remarkable success in crossing geographic borders and adapting to local environmental conditions. To investigate the potential role of microbial symbionts in these introductions, we examined the microbial communities of three ascidian species common in North Carolina harbors. Replicate samples of the globally introduced species Distaplia bermudensis, Polyandrocarpa anguinea, and P. zorritensis (n = 5), and ambient seawater (n = 4), were collected in Wrightsville Beach, NC. Microbial communities were characterized by next-generation (Illumina) sequencing of partial (V4) 16S rRNA gene sequences. Ascidians hosted diverse symbiont communities, consisting of 5,696 unique microbial OTUs (at 97% sequenced identity) from 47 bacterial and three archaeal phyla. Permutational multivariate analyses of variance revealed clear differentiation of ascidian symbionts compared to seawater bacterioplankton, and distinct microbial communities inhabiting each ascidian species. 103 universal core OTUs (present in all ascidian replicates) were identified, including taxa previously described in marine invertebrate microbiomes with possible links to ammonia-oxidization, denitrification, pathogenesis, and heavy-metal processing. These results suggest ascidian microbial symbionts exhibit a high degree of host-specificity, forming intimate associations that may contribute to host adaptation to new environments via expanded tolerance thresholds and enhanced holobiont function.