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1 1 2 3 2 Environmental heterogeneity and microbial inheritance influence - 4 5 6 3 associated bacterial composition of lamella 7 8 4 9 10 1 2* 1,3* 11 5 Charlotte Noyer , Emilio O. Casamayor , and Mikel A. Becerro 12 13 6 14 15 1 2 7 Department of Marine Biology, Department of Continental Ecology, Centro de Estudios Avanzados de Blanes 16 17 8 (CEAB-CSIC), Accés a la Cala St. Francesc 14, 17300 Blanes, Girona, and 3 Natural Products and Agrobiology 18 19 Institute (IPNA-CSIC), Avda. Astrofísico Francisco Sánchez 3, La Laguna, Tenerife 38206, Spain. 20 9 21 22 10 23 24 11 25 26 12 Keywords: DGGE, quantitative-PCR, bacteria, 16S rRNA gene, Spongia lamella, Porifera, 27 28 13 bath sponge 29 30 31 14 32 33 15 *E-mail corresponding authors: 34 35 16 Mikel Becerro 36 37 17 Emilio O Casamayor 38 39 18 40 41 42 19 43 44 20 Running title: Bacterial diversity in Spongia lamella 45 46 21 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 1 64 65 22 ABSTRACT 1 2 23 are important components of marine benthic communities. High-Microbial- 3 4 5 24 Abundance sponges host a large diversity of associated microbial assemblages. However, the 6 7 25 dynamics of such assemblages are still poorly known. In this study, we investigated whether 8 9 10 26 bacterial assemblages present in Spongia lamella remained constant or changed as a function 11 12 27 of the environment and life cycle. Sponges were collected in multiple locations and at 13 14 28 different times of the year in the western Mediterranean Sea and in nearby Atlantic Ocean to 15 16 17 29 cover heterogeneous environmental variability. Co-occurring adult sponges and offsprings 18 19 30 were compared at two of the sites. To explore the composition and abundance of the main 20 21 22 31 bacteria present in the sponge mesohyl, embryos, and larvae we applied both 16S rRNA gene- 23 24 32 Denaturing Gradient Gel Electrophoresis (DGGE) and sequencing of excised DGGE bands, 25 26 27 33 and quantitative Polymerase Chain Reactions (qPCR). On average, the overall core bacterial 28 29 34 assemblage showed over 60% similarity. The associated bacterial assemblages fingerprints 30 31 35 varied both within and between sponge populations, and the abundance of specific bacterial 32 33 34 36 taxa assessed by qPCR significantly differed among sponge populations and between adult 35 36 37 sponge and offsprings. Sequences showed between 92 to 100% identity to sequences 37 38 39 38 previously reported in Genbank, and all were affiliated with uncultured invertebrate bacterial 40 41 39 symbionts (mainly sponges). Sequences were mainly related to Chloroflexi and 42 43 44 40 Acidobacteria, and a few to Actinobacteria and Bacteroidetes. Additional populations may 45 46 41 have been present under detection limits. Overall, these results support that both ecological 47 48 42 and biological sponge features may shape the composition of endobiont bacterial communities 49 50 51 43 in S. lamella. 52 53 44 54 55 56 45 57 58 59 60 61 62 63 2 64 65 1 46 INTRODUCTION 2 3 4 47 A large proportion of marine organisms is hidden and yet undescribed, either as cryptic 5 6 48 species [1] or as symbionts [2,3]. A non-negligible part of the disregarded bacterial 7 8 49 communities is hidden both inside and on the surface of marine invertebrates, living as 9 10 11 50 associated or symbiotic partners [4-8]. That is particularly true for many marine sponges. 12 13 51 Microorganisms of the three domains of life [9-11] constitute up to 40% of the sponge 14 15 16 52 biomass [6, 12]. Bacteria seem to benefit from the stable and fertile environment provided 17 18 53 within sponge tissues (e.g., food supply, and specific ecological conditions including 19 20 21 54 protection against both UV and potential predators), and the composition of endobiont 22 23 55 bacterial communities would be shaped by ecological and biological sponge features, such as 24 25 56 environmental factors, habitat, health status, and life cycle among others. 26 27 28 57 29 30 58 The factors that structure sponge-bacteria associations remain however poorly understood. 31 32 33 59 It is now commonly admitted that sponge associated bacteria are both distinct from the free- 34 35 60 living marine bacterioplankton [11, 13-15], and sponge-specific [16] or even species-specific 36 37 38 61 [17]. Although some symbionts communities seemed first-sighted to form stable associations 39 40 62 across spatial and temporal scales [5, 16, 18], other studies have shown that microbial 41 42 43 63 communities can be affected by environmental changes: geographical variation [13, 19], 44 45 64 metal pollution [20], transfer into aquaculture [21, 22], or by physiological changes such as 46 47 65 disease outbreak [23]. Furthermore, host-specific factors are also believed to play a strong 48 49 50 66 structuring force shaping microbial communities as different sponge species from the same 51 52 67 habitat harbored distinct communities [24], and the same sponge species from the different 53 54 55 68 habitat showed similar communities [25]. Vertical transmission has been observed in various 56 57 69 marine sponges [7, 15, 26-29] and may also strongly shape symbionts composition explaining 58 59 60 70 the occurrence of sponge-specific bacterial clades in diverse hosts and locations [11]. 61 62 63 3 64 65 71 Regardless the mechanisms of bacterial transmission and acquisition, it is unclear whether 1 2 72 horizontally or vertically transmitted symbionts are equally relevant or whether they represent 3 4 5 73 the totality or a subsample of the microbes found in adult tissues. Comparing effects on 6 7 74 bacterial symbionts of both environment and host have not been performed yet but would 8 9 10 75 unravel factors that structure sponge microbial communities. Moreover, most of the studies 11 12 76 were carried out with a snapshot sampling strategy with limited replication [30, 31]. Limited 13 14 77 sampling design may hinder conclusions on the variability of sponge bacterial diversity since 15 16 17 78 it fails to properly address the ecological variation in sponge-associated microorganisms. 18 19 79 20 21 22 80 Populations of S. lamella (previously known as the Mediterranean S. agaricina, [32]) are 23 24 81 scattered throughout the Atlanto-Mediterranean region (e.g., [33]), where this species has 25 26 27 82 traditionally been collected as a bath sponge [32]. Although bacterial assemblages have been 28 29 83 reported as inhabitants of the genus Spongia [34], we know little about the microbial 30 31 84 consortium of S. lamella [35]. In the present study, we investigated bacterial symbionts from 32 33 34 85 sponges submitted to heterogeneous environmental conditions and collected in multiple 35 36 86 locations and at different times of the year in the western Mediterranean Sea and in nearby 37 38 39 87 Atlantic Ocean. Assuming that similarity between adults and offspring bacterial communities 40 41 88 is mainly consequence of vertical transmission, we also used a quantitative approach to 42 43 44 89 examine the main bacteria in adults, embryos, and larvae. This part of the study aimed at 45 46 90 gaining insight on the transfer and the effect of microbial inheritance on symbionts 47 48 91 communities, i.e., whether or not bacteria are randomly transferred to the next generation, and 49 50 51 92 whether or not embryos or larvae harbor the same bacterial communities than their parents. 52 53 93 Characterizing this variability is a critical step to unravel the distribution of associated 54 55 56 94 symbionts, sponge-bacteria interactions, and species-specific factor that shape symbionts 57 58 95 communities. 59 60 61 62 63 4 64 65 96 1 2 97 3 98 MATERIALS AND METHODS 4 5 99 6 7 100 Sampling. Spongia lamella (Demospongiae, , ) individuals 8 9 10 101 were collected by SCUBA diving in nine locations (Fig. 1) along the North West 11 12 102 Mediterranean and the Atlantic (see more details in [36]). In the area of Marseille (France), 13 14 103 specimens were gathered in Pharillon (43°12’N, 05°20’E), Plane (43°11'N, 05°23'E) and La 15 16 17 104 Ciotat (43°9'N, 05°35'E). In Catalonia (Spain), specimens were collected in Arenys (41°35'N, 18 19 105 02°33'E), and in Cap de Creus (42°17'N, 3°18'E). Populations were also collected in Cabrera 20 21 22 106 (39°7'N, 02°57'E; Balearic Islands, Spain), Ceuta (35°53'N, 05°18'W; Gibraltar area, Spain) 23 24 107 and in the Atlantic alongside Portugal (39°26'N, 9°30'W; Berlengas archipelago). Specimens 25 26 27 108 were collected along three years (2005-2007). When it was possible 10 specimens were 28 29 109 collected at each location. Three sponge specimens collected in Marseille in 2006 were 30 31 110 selected because of the presence of embryos, which gathered in clumps in brooding chambers 32 33 34 111 into the parent mesohyl (Fig. 2A), and were manually dissected. Visually, density of embryos 35 36 112 varied between individual sponges, however as no measurement was made, we could not 37 38 39 113 determine any correlation with sponge size. Three additional samples containing mature 40 41 114 larvae were collected from Els Bullents (Cala Canyelles, Lloret de Mar; 41º 42' N, 02º 52' E) 42 43 44 115 in July 2008. About 30 minutes after the transfer of the 3 sponges from Els Bullents into 45 46 116 individual aquaria, we observed the release of larvae from each of the tree sponges. Larvae of 47 48 117 S. lamella were oval and showed a black pigmented ring located at one of the larval poles, 49 50 51 118 surrounded by flagella which protrude outside. The other larval pole displayed a hummock 52 53 119 without any particular pigmentation (Fig. 2B). 54 55 56 120 57 58 59 60 61 62 63 5 64 65 121 Sponge tissues were transferred in plastic bags containing seawater and stored in a cooler with 1 2 122 ice until further processing (usually 2h after sampling). Samples were then rinsed and kept in 3 4 5 123 a series of absolute ethanol baths to prevent ethanol dilution by sponge water content and 6 7 124 sponge tissues modification. Sponge tissues were finally preserved in absolute ethanol (100 % 8 9 10 125 final concentration) and stored at –20°C until processed. 11 12 126 13 14 127 Extraction and amplification of bacterial DNA. DNA was extracted with the methodology 15 16 3 17 128 previously described [37] from sponge mesohyl tissue (around 4 mm ), embryos (a pool of 18 19 129 embryos from the same brooding chamber), and larvae (individually). DNA extracts were 20 21 22 130 dissolved in 50 µl of sterile autoclaved water and run in an agarose gel to check integrity and 23 24 131 concentration using a standard mass ladder (DNA Ladder, Bioron). Very small differences in 25 26 27 132 yield extraction were visualized among samples. Such differences were not expected to 28 29 133 produce qualitative changes in the DNA mixtures, and the results were normalized using 30 31 134 relative abundances in the fingerprinting analysis for an accurate inter-samples comparison. 32 33 34 135 DNA extracts were kept at –20°C until use. 35 36 136 A fragment (c. 600 bp) of the bacterial 16S rRNA gene was PCR amplified with 37 38 39 137 universal bacterial primers 341F (5’-CCT ACG GGA GGC AGC AG-3’) with a GC-clamp at 40 41 138 the 5’-end and 907RM (5’-CCG TCA ATT CMT TTG AGT TT-3’) [38]. The 50 µl PCR 42 43 44 139 mixture consisted of 80 ng of genomic DNA, 5µl of 10X Taq polymerase buffer, 0.75 µl of 45 46 140 MgCl2 (100µM), 1 µl of dNTP (10 mM each), 2.5 µl of each primers (10µM), 1.5µl of Bovine 47 48 141 Serum Albumin (BSA at 6mg/ml), and 1.25 U Taq polymerase (Boiron). Sigma pure Water 49 50 51 142 was used as negative control. Touchdown PCR was performed in a MWG Primus 52 53 143 thermocycler. The entire amplification program was 5 min at 94°C, 10 cycles of 1 min at 54 55 56 144 94°C, 1 min at 71°C (with 1°C decrease every cycle), 3 min at 72°C, 20 cycles of 1 min at 57 58 145 94°C, 1 min at 61°C, 3 min at 72°C and a final extension time of 3 min at 72°C. Then PCR 59 60 61 62 63 6 64 65 146 products were run in 1.5% agarose gels, stained in an aqueous ethidium bromide solution, and 1 2 147 visualized under UV next to a standard mass ladder (DNA Ladder, Boiron). 3 4 148 5 6 7 149 DGGE and 16S rRNA gene sequencing. DGGE analysis was run in a Bio-Rad Dcode 8 9 150 universal Mutation Detection System (BioRad) on 6% polyacrylamide gel in 1X TAE (40 10 11 12 151 mM Tris Base, 20 mM Sodium acetate trihidrate and 1 mM EDTA). We used a 40-75 % 13 14 152 vertical denaturant gradient (100 % denaturant agent is 7 M urea and 40 % deionized 15 16 153 formamide). Comparable amounts of PCR products (c. 600 ng DNA) were loaded for each 17 18 19 154 sample as reported [38]. Gels were run for 5 hours at 195 V at 60°C and stained with nucleic 20 21 155 acid stain SybrGold (Molecular Probes) solution (0.125 µl/ml) for 45 min, rinsed with MilliQ 22 23 24 156 water and photographed with Geldoc system (supplementary Fig.S1). Images of the gels were 25 26 157 analyzed using the Gels plot lanes tool of ImageJ software 1.38X (Wayne Rasband, National 27 28 29 158 Institutes of Health, USA). After background subtraction, the intensity of each band was 30 31 159 measured by integrating the area under the peak and was expressed as percentage of the total 32 33 160 intensity in the lane. This allowed measurement of the relative abundance of each band and 34 35 36 161 comparison of bacterial fingerprints among and within sponge populations. Bands with 37 38 162 intensities <1% of total intensity were excluded. 39 40 41 163 Prominent bands were excised from the gel under UV, resuspended in 25 µl of MilliQ 42 43 164 water and stored at 4ºC overnight. An aliquot (1-4 µl) of supernatant was used for PCR 44 45 46 165 reamplification with the original primer set, and the PCR product was sequenced using 47 48 166 external sequencing facilities (www.macrogen.com). Sequences were sent to BLAST search 49 50 51 167 (http://www.ncbi.nlm.nih.gov/BLAST/) to get a first indication of the sequence affiliations, 52 53 168 and to determine their closest relative in the database. Sequences were also inserted into the 54 55 169 optimized and validated tree available in ARB (Technical University of Munich, Munich, 56 57 58 170 Germany; www.arb-home.de) using the maximum parsimony criterion and a special ARB 59 60 171 parsimony tool that did not affect the initial tree topology to confirm BLAST affiliations. 16S 61 62 63 7 64 65 172 rRNA gene sequences were deposited in EMBL-GenBank under accession numbers 1 2 173 AM849589 to AM849614. 3 4 174 5 6 7 175 Quantitative-PCR assays. Quantitative PCR was used to quantify six bacterial clades, i.e., 8 9 176 Chloroflexi with one set of general GNSB941F-GNSB1340R [39, 40] and 2 sets of clade- 10 11 12 177 specific primers; Acidobacteria with 2 sets of clade specific primers, and Actinobacteria with 13 14 178 one set of primers. Primers from specific clades were designed from the bacterial sequences 15 16 179 obtained in a previous study (see more details in [35]). DNA of ten random specimens for 17 18 19 180 each of the eight populations (except for the population of Cap de Creus where only 8 20 21 181 specimens were found) was extracted from around 4 mm3 of mesohyl using DNAeasy Blood 22 23 24 182 and Tissue (Qiagen). We followed the manufacturer’s instructions except for a 5 min 25 26 183 incubation time before elution in a total volume of 75 µl supplied buffer. DNA concentrations 27 28 29 184 were then determined by Qubit® Quantitation (Invitrogen). The assays were conducted on a 30 31 185 Stratagene Mx3005P QPCR system. Each 25µl reaction contained 12.5 µl of 2x Brilliant 32 33 186 SYBR® Green QPCR Master Mix (Stratagene), 0.4 µM of each primer, 125 mM of BSA 34 35 36 187 (Promega), 30 nM of ROX reference dye, and 2 µl of template DNA at the optimal dilution. 37 38 188 PCR conditions were 10 min at 95°C, followed by 40 cycles of 95°C for 1 min, 30 s at 64°C 39 40 41 189 for the Acidobacteria runs and at 58°C for the other primers, and 72°C for 1 min, and finally 42 43 190 all PCR products were subjected to a melting curve analysis to verify the specificity of the 44 45 46 191 amplification. As various assays were run given the number of samples, we adjusted the 47 48 192 fluorescence threshold to 0.099 to allow the comparison of all of the runs and the 49 50 51 193 quantification with standard curves [35]. Adjusted threshold cycle (Ct) values were thus 52 53 194 averaged over triplicates for each sample. Negative control included three reactions without 54 55 195 DNA as control for contamination. Primer specificity was confirmed by running amplicons on 56 57 58 196 agarose gel and by melting curve analyses. Copy numbers per µg DNA were calculated from 59 60 197 the Ct value according to the standard curve previously obtained [35], the DNA 61 62 63 8 64 65 198 concentrations determined by Qubit® Quantification (Invitrogen) and the dilution applied. As 1 2 199 the six targeted bacterial groups achieved most of the bacterial assemblage, we calculated the 3 4 5 200 relative abundances as the ratio between the measured copy numbers for each group-specific 6 7 201 qPCR assay to the total copy number obtained for the 6 assays in each specimen. For the life- 8 9 10 202 stage comparison, only the 5 group-specific primers were used. 11 12 203 13 14 204 Statistical Analysis. We run statistical calculations available in the PRIMER v3.1 computer 15 16 17 205 program [41] to analyze differences in bacterial community as a function of sponge 18 19 206 population. Square root data were used to calculate Bray-Curtis similarity. Community 20 21 22 207 dissimilarities were visualized by cluster analysis using a single linkage clustering model and 23 24 208 non-metric multidimensional scaling (MDS) plots, and statistically tested by Analysis of 25 26 27 209 Similarity (ANOSIM). An exploratory similarity breakdown using the SIMPER procedure 28 29 210 was used to quantify the relative contribution of bacterial bands to dissimilarities, and the 30 31 211 pairwise bacterial dissimilarities between pair of sponge populations. 32 33 34 212 We compared quantitative differences in bacterial-clades proportion between the 35 36 213 sponge populations using MANOVA on rank-transformed clade relative abundances. We 37 38 39 214 used Post hoc Tukey HSD to test for differences between populations. We also used a 40 41 215 randomized block design multivariate analysis of variance (MANOVA) on rank transformed 42 43 44 216 data, to test whether clade proportions differ as function of life stages and sponge specimen, 45 46 217 both in Marseille (embryos vs. adults) and Els Bullents (larvae vs. adults). 47 48 218 49 50 51 219 RESULTS 52 53 220 As assembling data from different DGGE gels lead to significant gel effect (One way 54 55 56 221 ANOSIM, global R=0.725, p=0.001, data not shown), DGGE gels were analyzed individually 57 58 222 in all the pairwise combinations. The number of DGGE bands in the fingerprints was 59 60 61 62 63 9 64 65 223 relatively similar among samples (25-27 bands) and cluster analyses assigned sponge 1 2 224 specimens from the same population mostly into the same cluster (see two examples in Fig. 3, 3 4 5 225 where one specimen of Arenys clustered with Cap de Creus). Overall the core bacterial 6 7 226 assemblage for the 8 sponge populations analyzed showed over 60% similarity, being the 8 9 10 227 bacterial assemblage in Ceuta’s population the most heterogeneous (SIMPER analysis, 11 12 228 average similarity 71%), followed by Arenys, Cabrera, and Cap de Creus (78, 79, and 85%, 13 14 229 respectively). From the set of DGGE gels, we selected 26 bands for 16S rRNA gene 15 16 17 230 sequencing covering >85% of total band intensities. Sequences showed between 92 to 100% 18 19 231 identity to nucleotide sequences previously deposited in Genbank, and were all affiliated with 20 21 22 232 uncultured bacterial symbionts of invertebrates (mainly sponges). Overall, we found that 23 24 233 Chloroflexi (13 sequences) and Acidobacteria (11 sequences) phyla were the most abundant 25 26 27 234 groups in the DGGE gels. One sequence matched Actinobacteria (98% identity with 28 29 235 sequences obtained from the sponges Xestospongia muta and Ircinia felix), and the remaining 30 31 236 sequence was a Bacteroidetes, again, closely related to an uncultured Ircinia felix endobiont 32 33 34 237 (97% identity). Interestingly, several sequences were allocated in two distantly related clades 35 36 238 previously reported within Chloroflexi (CL-1 and CL-2, respectively) and within 37 38 39 239 Acidobacteria (AC-1 and AC-2, respectively). 40 41 240 42 43 44 241 Quantitative-PCR analysis of Spongia-associated bacteria. Using qPCR analysis, 45 46 242 we observed significant variations in the relative abundance of the main bacterial clades 47 48 243 among sites (MANOVA, Wilks' Lambda F=8.623, p<0.001; Fig. 4, and see absolute values in 49 50 51 244 supplementary Fig. S2). All sponge specimens showed the same proportion of Chloroflexi 52 53 245 using the general primers set (F=1.525; p=0.173; Fig. 4). However, we observed consistent 54 55 56 246 differences at the clade specific level. Thus, the Atlantic population was significantly more 57 58 247 enriched (relative abundance) in CL-1 than specimens from Arenys, Cabrera, and Ceuta (Fig. 59 60 61 62 63 10 64 65 248 4). Concerning the Acidobacteria, the clade AC-1 was more enriched in Arenys, Cap de 1 2 249 Creus, and Pharillon populations, and less abundant in the Atlantic, whereas AC-2 was more 3 4 5 250 homogeneously distributed. AC-2 was significantly more abundant in La Ciotat and less 6 7 251 abundant in the Atlantic (Fig. 4). Actinobacteria had a significantly higher relative abundance 8 9 10 252 in Portugal, Arenys, and Cap de Creus than in Cabrera (Fig.4). 11 12 253 13 14 254 Bacterial relative abundance in adults, embryos, and larvae. Significant 15 16 17 255 quantitative differences were observed in the bacterial community amplified by qPCR both 18 19 256 among life stages (adults vs. embryos, Wilks' Lambda F = 14.741, p<0.001, Fig. 5 and see 20 21 22 257 more details in supplementary Table S1). Univariate tests revealed that only the 23 24 258 Actinobacteria clade differed between the adults and their embryos (F = 36.957, p < 0.001). 25 26 27 259 In Els Bullents, we found significant quantitative differences in the bacterial community 28 29 260 amplified by qPCR among life stages (adults vs. larvae, Wilks' Lambda F = 10.829, p = 30 31 261 0.019), but not between sponge specimens. Univariate tests revealed that only the 32 33 34 262 Actinobacteria clade differed between the adults and their larvae (F = 11.270, p = 0.010). 35 36 263 We also detected quantitative differences in the adult bacterial community between 37 38 39 264 Marseille and Els Bullents (MANOVA, Wilks' Lambda F = 4.663, p = 0.044). Acidobacteria 40 41 265 clade 2 and Actinobacteria clade were responsible for the differences (Univariate tests, 42 43 44 266 Acidobacteria clade 2: F = 9.441, p = 0.012; Actinobacteria: F = 13.06, p = 0.005, Fig. 5). 45 46 267 47 48 49 50 268 DISCUSSION 51 52 269 Until quite recently, sponge associated microorganisms were believed to form stable 53 54 55 270 communities across spatial and temporal scales [16, 18, 30]. However, more recent studies 56 57 271 have challenged the uniformity and stability of sponge- symbiont associations. Lack of 58 59 60 272 replication and/or absence of relative quantitative comparison may have contributed to 61 62 63 11 64 65 273 underestimate variability and thus diversity of bacteria within sponges. Most sequences 1 2 274 available in bacterial genes libraries were obtained from 1 to at most 3 individuals of each 3 4 5 275 species or locations. Nowadays, several studies show evidence that ecophysiological and 6 7 276 environmental factors may also influence bacterial variability [17, 20, 23]. Although we found 8 9 10 277 a common pool of bacteria inhabiting S. lamella (assemblages similarity, c.a. 60%), we also 11 12 278 observed significant variations in the DGGE fingerprints. Although sponge specimens in a 13 14 279 population clustered together according to their bacterial composition, we highlighted 15 16 17 280 variations among the bacterial assemblages both within and among sponge populations. 18 19 281 20 21 22 282 Despite some well-known limitations inherent to the DGGE method and its detection 23 24 283 limits [38, 42], this culture independent approach offers still a good compromise for the rapid 25 26 27 284 detection and identification of predominant microbial species and simultaneous comparison 28 29 285 among various samples. However, inter-gel comparison can be difficult [18, 19] especially 30 31 286 dealing with highly diverse samples. In our study, gels were individually compared to avoid 32 33 34 287 misinterpretation due to gel effects. Most studies dealing with bacterial diversity in sponges 35 36 288 using DGGE have focused on presence/absence data. Qualitative estimations are appropriate 37 38 39 289 for assemblage comparison from several host species [31, 43], but might underestimate 40 41 290 variation when comparing specimens of the same host species. Signal intensity of DGGE 42 43 44 291 bands can be applied to provide a rough overview of relative abundance changes from 45 46 292 predominant bacterial groups [38]. We were cautious in the number of PCR cycles run to 47 48 293 minimize the "plateau" phase and all samples were run under the same PCR and DGGE 49 50 51 294 conditions. Thus, if there was any PCR bias it should be the same in all lanes and samples, 52 53 295 and therefore comparison among samples is still valid. We were well aware that we worked 54 55 56 296 with normalized relative abundances for comparison among samples, and not with total 57 58 297 abundances. However, the recent application of Next Generation Sequencing approaches are 59 60 61 62 63 12 64 65 298 significantly expanding the knowledge in bacterial/invertebrates interactions (e.g. [44, 45]), 1 2 299 and will overcome some of the known limitations of fingerprinting methods. 3 4 5 300 6 7 301 Sequences retrieved from the DGGE gels indicated that they were all related to 8 9 10 302 uncultured bacteria and distantly related to the typical planktonic counterparts commonly 11 12 303 found in previous studies reported in the literature [e.g., 46]. Bacteria inhabiting S. lamella 13 14 304 resembled those in high-microbial-abundance sponges previously analyzed [11]. Most 15 16 17 305 sequences belonged to Chloroflexi and Acidobacteria. These two phyla and Actinobacteria 18 19 306 are the groups most frequently retrieved from 16S rRNA gene surveys in sponges [6] among 20 21 22 307 the 15 bacterial phyla so far identified [11]. Within the Chloroflexi and Acidobacteria, we 23 24 308 observed two distantly related clades within the same phylum specifically containing marine 25 26 27 309 sponge sequences. Acidobacteria clade 1 gathered together bacterial assemblages from Ircina 28 29 310 felix and Smenospongia aurea [7, 29]; whereas Acidobacteria clade 2 included sequences 30 31 311 from Aplysina fulva, Xestospongia testudinari, and Desmacidon sp. [14, 46]. Chloroflexi 32 33 34 312 clade 1 contained bacterial sequences from S. lamella, and from various sponge species such 35 36 313 as Ircina and Aplysina [6, 7, 14, 16, 21]. The Chloroflexi clade 2 contained also bacterial 37 38 39 314 sequences retrieved from diverse sponge species [11, 15, 29]. Interestingly, sequences excised 40 41 315 at the same position in the gel and sequenced to confirm they were identical had also 100% 42 43 44 316 identity to sequences obtained from Aplysina aerophoba collected in the same area in the NW 45 46 317 Mediterranean [48], and 99% identity to a sequence from Ancorina alata collected in 47 48 318 Northeastern New Zealand [49]. Further phylogenetic data with longer sequences may 49 50 51 319 confirm whether it corresponds to the same symbiotic Chloroflexi species. 52 53 320 The sequence affiliated to Actinobacteria grouped within a sponge-specific cluster 54 55 56 321 including Actinobacteria sequences from sponges collected in several oceans (Ircina felix, 57 58 322 Xetospongia muta, Xestospongia, testudinari, Aplysina aerophoba, Svenzea zeai, and 59 60 61 62 63 13 64 65 323 Ancoriana alata). Sequence 6d11 (AM849590) was closely related to Bacteroidetes 1 2 324 sequences stemmed from marine sponges (Geodia barretti, Ircinia felix I. strobilina, 3 4 5 325 Desmacidon sp., and Ancoriana alata,), and sequences from this phylum have been recently 6 7 326 retrieved from various sponge species, although clear phylogenetic affiliation remains still 8 9 10 327 ambiguous. Longer 16S rRNA gene fragments and powerful treeing methods are needed to 11 12 328 properly allocate this group. 13 14 329 15 16 17 330 In agreement with previous data [35, 50] we observed by qPCR analyses that 18 19 331 Chloroflexi was the most abundant bacterial phylum within the sponge mesohyl. Relative 20 21 22 332 abundance of Chloroflexi amplified by general primers seemed relatively stable in all sponge 23 24 333 populations. However, relative abundance of Chloroflexi CL-1 and CL-2 varied significantly 25 26 27 334 between sponge populations and mainly between Mediterranean and Atlantic populations. 28 29 335 These two specific primers did not cover the large abundance of Chloroflexi obtained with the 30 31 336 general primers, suggesting a larger diversity of Chloroflexi in S. lamella than in other sponge 32 33 34 337 genera [35]. Certainly, additional Chloroflexi groups not detected by DGGE excised bands 35 36 338 may inhabit the sponge mesohyl and deserve further investigations. Portugal population 37 38 39 339 displayed significant differences, harboring significantly more CL-1 and less CL-2, whereas 40 41 340 proportions of these 2 clades were similar in the remaining populations. Portugal also showed 42 43 44 341 significantly less Acidobacteria and more Actinobacteria compared to other populations. 45 46 342 Portugal population was the only sample obtained both from the Atlantic Sea and inside a 47 48 343 marine cave. Unfortunately we cannot split the two effects with the current experimental 49 50 51 344 design and any effect these features may have on the sponge bacterial composition remains to 52 53 345 be determined. In fact, it is well-known that changes in the host physiology (such as transfer 54 55 56 346 in aquarium and cultivation under artificial conditions, or disease outbreaks, [21-23] or abiotic 57 58 347 factors such as environmental stress associated with metal pollution or light [20, 50] can 59 60 61 62 63 14 64 65 348 modify microbial assemblages. Larger bacterial diversity in stressed or sick sponges may be 1 2 349 linked to a greater diversity of ecological niches available for bacteria and an increase of 3 4 5 350 nutrients following sponge cells decay [23]. 6 7 351 8 9 10 352 In this study, we also described the presence of embryos and larvae and compared the 11 12 353 relative abundances of main bacterial groups between different life stages of the sponge using 13 14 354 qPCR analyses. We provided information on the proportions of Chloroflexi, Acidobacteria 15 16 17 355 and Actinobacteria found in Spongia lamella and we hypothesized they were mainly 18 19 356 vertically transmitted to the next generation, as it has been reported in several sponge species 20 21 22 357 [7, 15]. We observed both similarities and differences in bacterial community between adult 23 24 358 sponges and their relative offsprings suggesting complex pathways for symbiont transmission 25 26 27 359 as previously reported in Hippiospongia and Spongia species [26, 52]. In brooding sponges, 28 29 360 nurse cells charged with endosymbiotic microbes engulfed from the mesohyl establish 30 31 361 cytoplasmic bridges with the zygote membrane, transferring microorganisms and nutrients. 32 33 34 362 When zygotes became embryos, they developed a cell follicle though also kept connected to 35 36 363 the surrounding tissue by a system of radiating mesohyl bridges. These bridges probably 37 38 39 364 facilitate the anchoring and/or feeding of the embryos during development and also allow 40 41 365 symbiotic bacteria to migrate from the adult mesohyl to the intercellular spaces of the 42 43 44 366 embryos [26]. Sponge adults and their respective embryos and larvae harbored the same 45 46 367 relative proportions of Chloroflexi and Acidobacteria clades. However, the proportion of 47 48 368 Actinobacteria was significantly higher in both embryos and larvae than in their respective 49 50 51 369 parents. If a portion of the microbial community migrated from the adult mesohyl to embryos, 52 53 370 higher proportion of Acidobacteria could simply be the result of a faster cell multiplication. 54 55 56 371 Once the larvae settled, environmental conditions affecting both host fitness and microbial 57 58 372 assemblage may balance relative abundances of the different populations [24]. 59 60 61 62 63 15 64 65 373 Parenchymella larva, the most common larval type in demospongiae, is commonly 1 2 374 classified among the lecitotrophic (non-feeding) meroplankton with limited swimming 3 4 5 375 abilities [53]. They are self-sustained by using stored material such as lipid inclusions or 6 7 376 bacteria phagocytosis [54]. Several studies highlight the importance of bacteria stored into 8 9 10 377 larvae as food source [26, 52, 54]. In contrast in Halisarca dujardini johnston there is no 11 12 378 evidence of lysis or digestion of bacteria at any stage of the development, although symbiotic 13 14 379 bacteria were present intercellularly in cleaving embryos and larvae [27]. Furthermore, 15 16 17 380 brooded larvae that develop using yolk (lecitotrophs) are typically larger, easier to see and 18 19 381 lack morphological characteristics that decrease fish predation compared to longer-lived 20 21 22 382 planktotrophic larvae. Brooded larvae of many diverse sessile invertebrates are commonly 23 24 383 distasteful to benthic fishes, and their unpalatability has often been attributed to chemical 25 26 27 384 defenses [55]. Among bacteria, Actinobacteria are known as one of the richest sources of 28 29 385 active secondary metabolites [56-60]. Thus, the presence of Actinobacteria in the embryos 30 31 386 and larvae might serve either as food source for the S. lamella lecitotrophic larva or as 32 33 34 387 chemical defense against potential predators, competitors, foulers, or infectious 35 36 388 microorganisms. 37 38 39 389 40 41 390 Overall, although the final causes for the variability found in this study remain 42 43 44 391 unclear, some assumptions can be set out, and a combination of horizontal (environmental) 45 46 392 and vertical (parental) transmission may be considered [11, 61]. A fraction of bacteria 47 48 393 environmentally transmitted may result from “selective enrichment” of specific bacteria that 49 50 51 394 remained undetectable in the surrounding plankton [16], and sponges must provide a 52 53 395 favorable habitat for them to grow over the methodological detection thresholds. Differences 54 55 56 396 between life stages may also be the result of transient bacteria acquired from the surrounding 57 58 397 water, or by a differential and preferential bacterial transmission. Competition and selection, 59 60 61 62 63 16 64 65 398 which are dynamic processes, affect the structure of the microbiota [62] and could also 1 2 399 explain variability in bacteria associated to sponge. Genetic variability of sponge populations 3 4 5 400 could also contribute to the observed variability in bacterial communities [24]. As most 6 7 401 bacterial groups appeared consistently through S. lamella populations and main bacterial 8 9 10 402 groups were present in sponge populations, a combination of “filtration-concentration” (which 11 12 403 depend on sponge physiology and environmental factors and are more proned to variations) 13 14 404 and “hereditary-colonization” (linked to true symbionts at the origin of the uniformity among 15 16 17 405 sponge associated bacteria) may be possibly accounting. These results might correspond with 18 19 406 multiple levels of specificity through which bacteria are associated to sponges, and should be 20 21 22 407 carefully considered for a more accurate understanding of sponge microbial ecology. 23 24 408 25 26 27 28 409 ACKNOWLEDGMENTS 29 30 410 We thank Thierry Perez for providing samples from the area of Marseille, Joanna Xavier for 31 32 33 411 the sponge specimens from Portugal, and Josep Carreras, Andrea Blanquer, and Xavier Turon 34 35 412 for help during field sampling. We also thank Alastair Hamilton for technical support during 36 37 38 413 the qPCR experiments at Integrin Advanced Biosystem. 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Dethlefsen L, McFall-Ngai M, Relman DA (2007) An ecological and evolutionary perspective 27 28 577 on human-microbe mutualism and disease Nature 449: 811-818 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 22 64 65 578 Figure Legends 1 2 579 3 4 580 FIG. 1: Locations of Spongia lamella sampling sites in NW Mediterranean Sea and Atlantic 5 6 581 Ocean. A zoom on the area of Marseille is shown. 7 8 582 9 10 583 FIG. 2: Spongia lamella life stages including (a) brooded embryos gathered within the parent 11 12 584 sponge (bar: 0.5 cm), as: aquiferous system; bc: brooding chamber, emb: embryos from 13 14 585 distinct brooding chambers into the adult mesohyl; and (b) newly released parenchymella 15 586 larva observed under light microscopy (bar: 500µm). 16 17 587 18 19 588 FIG. 3: Cluster diagrams of DGGE fingerprints of bacterial assemblages in Spongia lamella at 20 21 589 (a) local and (b) regional scales. Six sponge specimens were analyzed per site. 22 23 590 24 25 591 FIG. 4: Relative abundance as estimated by qPCR analysis of Chloroflexi (GNSB-941F, Cl1, 26 592 and Cl2), Acidobacteria (Ac1 and Ac2), and Actinobacteria (Actino) in several populations of 27 28 593 Spongia lamella. Vertical bars are standard errors (n=10 samples per population except Cap 29 30 594 de Creus n=8). ANOVA on rank-transformed proportion of clade relative abundance, p- 31 32 595 values and Post hoc Tukey HSD included. 33 34 596 35 36 597 FIG. 5: Relative proportion as estimated by qPCR analysis of Chloroflexi (clades Cl1, and 37 598 Cl2), Acidobacteria (clades Ac1 and Ac2), and Actinobacteria (clade Actino); in Spongia 38 39 599 lamella, from adults (A_emb) and embryos (emb) sampled in Marseille; and adults (A_larv) 40 41 600 and larvae (larv) sampled in Els Bullents. Vertical bars are standard errors. P-value from the 42 43 601 randomized block (Multivariate) Analysis of Variance included. NS: non significant. 44 45 602 46 47 603 Supplementary figures 48 604 Fig. S1. Negative images of DGGE gels obtained in this study 49 50 605 51 52 606 Fig. S2. Abundance as estimated by qPCR analysis of six bacterial clades in several 53 54 607 populations of Spongia lamella, i.e., Chloroflexi (GNSB-941F, Cl1, and Cl2), Acidobacteria 55 56 608 (Ac1 and Ac2), and Actinobacteria (Actino). 57 609 58 59 60 61 62 63 23 64 65 Figure Figure Figure Figure Figure