During the central and eastern Torres Strait survey in November 2006, tissue samples of 10 individuals of the sponge Coscinoderma matthewsi were collected from 5 island groups: Ugar (Stephen Island) and Erub (Darnley Island) in eastern Torres Strait; and the Masig group (Kodall Island and Keats Island), Poruma (Coconut Island) and Warraber (Sue Island) in central Torres Strait. These island groups are on average, 66 km apart. All sponge samples were placed in separate cryo-tubes and preserved in liquid nitrogen until they could be stored at -80°C. Approximately 2 g of tissue from each sample was homogenised in liquid nitrogen and 750 µl of lysis buffer [100 mM Tris pH 9, 100 mM EDTA, 1% SDS, 100 mM NaCl, 0.5 mg/ml Proteinase K], and subsequently incubated at 65°C for 1 hour with gentle agitation. KoAc was added to a final concentration of 1 M, followed by incubation on ice for 30 minutes. The samples were centrifuged at 8000 rpm for 15 minutes, and the supernatant was reserved for DNA precipitation with isopropanol using the standard protocol.A fragment of nuclear DNA containing part of the 28S rRNA gene was amplified for all individuals using RD3A (5'-GACCCGTCTTGAAACACGA) and RD5B2 (5'- ACACACTCCTTAGCGGA) primers. Recombinant Pfu Polymerase (Fermentas) was used for the PCR. A total of 50 µl of reaction mixture was prepared for each sample according to the protocol. PCR was performed under the following conditions: initial denaturation at 95°C for 3 minutes; 35 cycles of 95°C for 30 seconds, 50°C for 20 seconds, 72°C for 1 minute; a final extension step of 72°C for 10 minutes. Products were purified with QIAquick (Qiagen) columns according to protocol. Sequencing was performed at Macrogen Inc. with a 3730xl DNA analyser using both forward and reverse primers.For microbial analysis, a DNA fingerprinting technique (denaturing gradient gel electropohoresis - DGGE) was used to determine the stability of bacterial associations within Coscinoderma matthewsi across wide spatial scales.Four replicate sponges were analysed from Keats Island and Kodall Island and three replicate sponges were analysed from Erub, Ugar, Poruma and Warraber. DNA was extracted from individual sponges by homogenising approx 1g of tissue from each individual in 0.5 ml of grinding buffer (2 ml 1 M Tris, 4 ml 0.5M EDTA, 2 ml 10% SDS, 400 µl 5 M NaCl and 11.6 ml distilled water). Tubes were immersed in liquid nitrogen and ground with plastic pestles. Samples were incubated at 65ºC for 60 min prior to addition of 187 µl 5 M potassium acetate. Samples were incubated on ice for 30 min and centrifuged at 8000 x g for 15 min. The supernatants were transferred to fresh tubes and DNA was precipitated with 0.8 vol of isopropanol.The 16S rDNA from each sample was amplified by PCR with universal bacterial primers 1055f: 5'-ATG GCT GTC GTC AGC T-3' and 1406r: 5'-ACG GGC GGT GTG TAC-3'. The reverse primer was modified to incorporate a 40 bp GC clamp. Primers 1055f and 1406r match over 56,000 and 62,800 sequences respectively in the Ribosomal Database Project. Products from triplicate PCR reactions were combined and 15 µl applied to duplicate 40% wt/vol polacrylamide (37:5:1) gels containing a 50-70% denaturing gradient of formamide and urea. Gels were electrophoresed at 60ºC for 17 h in 1 x TAE buffer at 50V using the Ingeny D-Code system. Gels were stained with 1 x Sybr Gold for 30 min, visualised under UV illumination and photographed.
