Supplementary Data- BIOLIEF #7

Supplementary Data- BIOLIEF #7

Table S1. Sample size, individual size range and average values for the parameters used for statistical analyses (GLM ANOVA) presented in Table 3 and the Results Section.These data were used for the following comparisons:(a) flesh dry weight according to infestation level; (b) brood dry weight according to infestation level (0, 1, 2, 3) and brood developmental stage; (c) number of capsules per brood according to presence vs. absence of Cliona celata (level 0 vs. level 3); (d) number of embryos per capsule according to infestation.

(a)Flesh dry weight

(b)Brood dry weight (Brood developmental stage: y= young, i= intermediate, m=mature)

(c) Number of capsules per brood(d) Number of embryos per capsule

Supplementary Data – Biolief #7

Appendix - Molecular identification of the boring sponge Cliona celata

Based on tissue colour, excavation characteristics and spicule morphology, we unambiguously identified the endolithic sponge sampled on C. fornicata shells as a member of the Clionidae (Hayward & Ryland 1996; Rützler 2002). However, the species identity of clionidsponges isparticularly difficult to determine usingonly on morphological criteria due to the paucity of taxonomic characters (Fromont et al. 2005). Barrucca et al. (2007) showed that the D2 region of 28S rDNA is a goodmarker for identifying specimens to the species level: they observed a maximal divergence of 14.98% by sequencing 13 Cliona sponge species. Molecular analyses based on this D2 region were thus carried out to confirm the specific status of the boring sponge observed in the study population of Crepidula fornicata, namely Cliona celata.

Sponges corresponding to α and β (i.e. intermediate stage where the sponge outgrows the gallery and starts to cover the shell) stages were sampled froma C. fornicata specimen; one individual at the γ (‘free-living’) stage was sampled in the Bay of Morlaix by the divers atthe Roscoff Biological Station. Total DNA was extracted using a conventional phenol-chloroform protocol (Sambrook and Russell 2001) and checked on a 1% agarose gel. Following protocols and primer sequences reportedin Barucca et al. (2007), we sequenced a ~630bp fragment of D2 region of 28S rDNA using primers 5’-AAGGTGAAAAGTACTTTGAAAAGA-3’ and 5’-TCCGTGTTTCAAGACGGGTC-3’. Sequences were obtained using the Big Dye Terminator v.3.1 Cycle Sequencing kit on an ABI 3130 XL DNA sequencer following the manufacturer’s protocol (Applied Biosystems). Sequences were edited in BioEdit version 5.0.1 (Hall 1999), aligned with Clustal V (Jeanmougin et al. 1998) and compared with sequences available in GenBank using BLAST software ( including the sequences from Barucca et al. (2007).

Comparison of approximately 435 base pairs of the D2 sequenced region of 28S rDNA with sequence from C. celata published by Barucca et al. (2007) (GenBank accessions nos.AM293628, AM293629 and AM293630) showed from91 to 99% similarity. The alignments of these sequences are presented in the figure below. This molecular analysisthus confirmed that the boring sponge sampled on C. fornicatashells in the Bay of Morlaixare indeed C. celata specimens.

References:

Barucca M, Azzini F, Bavestrello G, Biscotti M, Calcinai B, Canapa A, Cerrano C and Olmo E (2007) The systematic position of some boring sponges (Demospongiae, Hadromerida) studied by molecular analysis. Mar Biol. 151: 529-535. Doi: 10.1007/s00227-006-0486-y

Fromont J, Craig R, Rawlinson L and Alder J (2005) Excavating sponges that are destructive to farmed pearl oysters in Western and Northern Australia. Aquac Res. 36: 150-162. Doi: 10.1111/j.1365-2109.2004.01198.x

Rützler K (2002) Family Clionaidae D'Orbigny, 1851. In: Hooper J and Van Soest R Sistema Porifera: a guide to the classification of sponges, Academic Plenum New York, 173-185

Hayward PJ and Ryland JS (1996) Handbook of the marine fauna of North-West Europe. OxfordUniversity Press, Oxford, UK

Figure Appendix - Alignment of nucleotide sequences fromthe D2 region of 28S rDNA of Cliona celata

alphaD2, betaD2 and gammaD2 are the samples analysed in this study. Other sequences were retrieved from GenBank.