Quantitative comparison of bacterial communities in two Mediterranean sponges

Abstract

Marine sponges can host in their tissues abundant and diverse bacterial communities. Lack of truly quantitative data on bacterial abundance and dynamics limits our understanding of the organization and functioning of these endobiotic communities. In this technical note, we describe a quantitative polymerase chain reaction approach to quantify the relative abundance of multiple clades of three major sponge-associated bacterial phyla: Chloroflexi, Acidobacteria, and Actinobacteria. To test our approach we used the Mediterranean sponges Spongia lamella and Aplysina aerophoba. We designed five out of the six primer sets used in our study. We tested the new primer sets for specificity and optimized their conditions. Our preliminary data showed that Spongia lamella had larger bacterial abundance than Aplysina aerophoba, except for one clade of Chloroflexi. The two Chloroflexi clades investigated in our study amplified a fraction of the Chloroflexi present in Spongia lamella and most of what is present in Aplysina aerophoba, suggesting a more diverse Chloroflexi population in Spongia lamella than in Aplysina aerophoba. This quantitative technique has a great potential to provide a rapid and robust assessment of sponge microbial target and could contribute to deciphering the complexity of these largely unknown host-symbiont interactions.

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

“…Our calculated gene numbers (2.35 × 10 7 –2.18 × 10 8 for HMA and 3.09 × 10 4 –7.89 × 10 7 for LMA) for this taxon are in the range as has been reported previously (Cassler et al ., ; Abdelmohsen et al ., ). Another publication showed much lower actinobacterial gene copy numbers in sponges (Noyer et al ., ) which is not surprising given that the utilized primer pair showed only 2% phylum‐level coverage. In terms of bioprospecting, the presented qPCR data demonstrate that marine sponges are far better sources than seawater for actinobacterial isolation work as smaller volumes (7.5–20 mg) are needed to extract the same amount of genomic DNA.…”

“…Only primers that showed specificities > 90%, that is, < 5/48 clones with nontarget sequence, were used for the present study. Previously published primer combinations for analyzing microbial consortia of sponges (Noyer et al ., ) and additional qPCR primers for taxon‐specific quantification (Bacchetti De Gregoris et al ., ) did not meet the desired specificities and/or reaction efficiencies and were excluded from this study.…”

Relationship between genetic, chemical, and bacterial diversity in the Atlanto-Mediterranean bath sponge Spongia lamella

“…Individuals were typed at seven microsatellite loci, genotyping protocols are described in Noyer et al (2009Noyer et al ( , 2010. For each individual, we measured (1) MLH calculated as the proportion of loci that were heterozygous corrected for non-scored loci (Lesbarreres et al, 2007;Chapman et al, 2009;Pujolar et al, 2009) and (2) mean d 2 measured by LD = log (mean d 2 ?…”

“…We analyzed images of the gels using the Gels plot lanes tool of ImageJ software 1.38X (Wayne Rasband, National Institutes of Health, USA) according to Noyer (2010) and references therein. To perform quantitative analyses using qPCR, we used five specific primer pairs designed in Noyer et al (2010), to amplify and quantify specifically bacterial clades using a Stratagene Mx3005P QPCR system and 29 Brilliant SYBR Ò Green QPCR Master Mix (Stratagene) (Noyer, 2010). For each individual screened, we used Shannon diversity index from the DIVERSE procedure available in PRIMER 6 (Clarke & Warwick, 2001).…”

Patterns of Chemical Diversity in the Mediterranean Sponge Spongia lamella

“…Images of the gels were analyzed using the Gels plot lanes tool of ImageJ software 1.38× (Wayne Rasband, National Institutes of Health, USA). After background subtraction, the intensity of each band was measured by integrating the area under the peak and was expressed as percentage of the total intensity in the lane [38], [66]. We measured the relative abundance of each band and compared bacterial profiles among and within sponge populations.…”