Quantification of bacterial and archaeal symbionts in high and low microbial abundance sponges using real-time PCR

RESEARCH ARTICLE Quantification of bacterial and archaeal symbionts in high and low microbial abundance sponges using real-time PCR Kristina Bayer, Janine Kamke & Ute Hentschel Department of Botany II, Julius-von-Sachs Institute for Biological Sciences, University of Wuerzburg, Wuerzburg, Germany Present address: Janine Kamke, AgResearch Ltd, Grasslands Research Centre, Tennent Drive Private Bag 11008, Palmerston North 4442, New Zealand MICROBIOLOGY ECOLOGY Received 14 April 2014; revised 6 June 2014; accepted 10 June 2014. Final version published online 9 July 2014. DOI: 10.1111/1574-6941.12369 Editor: Gary King Abstract In spite of considerable insights into the microbial diversity of marine sponges, quantitative information on microbial abundances and community composition remains scarce. Here, we established qPCR assays for the specific quantification of four bacterial phyla of representative sponge symbionts as well as the kingdoms Eubacteria and Archaea. We could show that the 16S rRNA gene numbers of Archaea, Chloroflexi, and the candidate phylum Poribacteria were 4–6 orders of magnitude higher in high microbial abundance (HMA) than in low microbial abundance (LMA) sponges and that actinobacterial 16S rRNA gene numbers were 1–2 orders higher in HMA over LMA sponges, while those for Cyanobacteria were stable between HMA and LMA sponges. Fluorescence in situ hybridization of Aplysina aerophoba tissue sections confirmed the numerical dominance of Chloroflexi, which was followed by Poribacteria. Archaeal and actinobacterial cells were detected in much lower numbers. By use of fluorescence-activated cell sorting as a primer- and probe-independent approach, the dominance of Chloroflexi, Proteobacteria, and Poribacteria in A. aerophoba was confirmed. Our study provides new quantitative insights into the microbiology of sponges and contributes to a better understanding of the HMA/LMA dichotomy. Keywords marine sponges; microbial symbionts; quantitative real-time PCR; fluorescence in situ hybridization; single-cell sorting. Introduction Marine sponges (phylum Porifera) are sessile animals with a fossil record dating back to the Precambrian times (Li et al., 1998). These animals are highly effective filter feeders and ecologically important reef builders in marine ecosystems. Sponges are commonly classified as ‘high microbial abundance’ (HMA) or ‘low microbial abundance’ (LMA) sponges (Reiswig, 1981; Hentschel et al., 2003). HMA sponges harbor highly diverse and dense microbial consortia within their tissues which can contribute up to 35% of the animal’s biomass (Hentschel et al., 2012). Representatives of at least 29 bacterial phyla (i.e. Actinobacteria, Acidobacteria, Chloroflexi, Cyanobacteria, Bacteriodetes, Nitrospirae) including several candidate phyla (i.e. candidate phylum FEMS Microbiol Ecol 89 (2014) 679–690 Poribacteria, candidate phylum Tectomicrobia) (Fieseler et al., 2004; Wilson et al., 2014) and two archaeal lineages have been identified in HMA sponge species so far (Webster et al., 2010; Lee et al., 2011; Schmitt et al., 2012a, b; Moitinho-Silva et al., 2014). On the other hand, the mesohyl matrix of LMA sponges is virtually devoid of microorganisms, as judged by electron microscopical inspection (Vacelet & Donadey, 1977; Weisz et al., 2007; Giles et al., 2012; Gloeckner et al., 2013; Poppell et al., 2013). Phylogenetic analysis revealed that Cyanobacteria and Proteobacteria (Alpha-, Beta-, or Gamma-) are the dominant phyla in LMA sponges (Erwin et al., 2011; Giles et al., 2012; Blanquer et al., 2013; Moitinho-Silva et al., 2014). It was further shown that at least one LMA sponge, Stylissa carteri from the Red Sea, harbors its own distinct microbiota and is not ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved Correspondence: Kristina Bayer, Department of Botany II, Julius-von-Sachs Institute for Biological Sciences, University of Wuerzburg, Julius-von-Sachs Platz 3, 97082 Wuerzburg, Germany. Tel.: +49 931 31 86217; fax: +49 931 31 86235; e-mail: 680 ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved Materials and methods Sample collection and processing The Mediterranean sponges A. aerophoba and Dysidea avara were collected in May 2012 and C. reniformis in September 2009 by SCUBA diving at 5–12 m depth offshore Rovinj, Croatia (45°090 N, 13°630 E). The Red Sea sponges S. carteri and Xestospongia testudinaria were collected by SCUBA diving at Fsar reef (22°230 N; 39°030 E) at depths of 13–15 m off the coast of Thuwal, Saudi Arabia, in January 2012. The sponges Xestospongia muta, Ircinia felix, and Callyspongia vaginalis were collected offshore Little San Salvador Island, Caribbean Sea, Bahamas (24°340 N; 75°580 W) at 12 m depth in July 2003. Three biological replicates per species were sampled. Small tissue pieces were removed with a sterile scalpel, rinsed in 0.2-lm filtered seawater, immediately frozen in liquid nitrogen, and stored at
80 °C until use. For seawater collections, 7–10 L of Mediterranean and Red Sea seawater was filtered using 0.2-lm-pore-size bottle top SFCA membrane filters (Nalgene, Germany). Sediment samples were collected from the Bahamas location at a depth of 3 m. Seawater filters and sediment samples were stored at
80 °C until further use. Extraction and quantification of genomic DNA Frozen sponge samples were ground to fine powder with liquid nitrogen using a sterile mortal and pistil. An effort was made to cut sponge pieces so that the natural mesohyl vs. pinacoderm proportions were retained. Genomic DNA from sponge powder, sediment samples, and seawater filters were then extracted using the Lysing matrix E tubes (MP Biomedicals, Illkirch, France) and the AllPrep DNA/RNA Mini Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. The extracted DNAs were checked for quality using the NanoDrop C2000 instrument (Peqlab, Erlangen, Germany). For accurate quantification of the genomic DNA, the Qubit 2.0 Fluorometer and Qubit dsDNA BR and HS Assay kits (Invitrogen, Darmstadt, Germany) were used. Genomic DNA was stored at
20 °C until further use. Evaluation of primer specificities All primers used in this study are listed in Table 1. Specificities of the phylum-level primers Actinobacteria, Poribacteria, Cyanobacteria, Chloroflexi, and those for the domain Archaea were tested by PCR using metagenomic DNA from A. aerophoba as template. Primer specificity for the phylum Proteobacteria could not be confirmed, FEMS Microbiol Ecol 89 (2014) 679–690 merely a reflection of seawater (Moitinho-Silva et al., 2014). While the microbial diversity of marine sponges is well investigated today, quantitative information on microbial abundances in mesohyl tissues remains scarce. Based on transmission electron microscopical observations, Vacelet (1975) estimated that 37% of the Aplysin (...truncated)