Population genetics and microbial analysis of the Dictyoceratida sponge, Coscinoderma matthewsi, from central and eastern Torres Strait, Australia (MTSRF Project 1.3.2)
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.\n This project was undertaken to determine connections between sponge populations in central and eastern Torres Strait and to assess whether there would be any risks associated with translocation of sponges across large areas. Risks investigated were the possibility of transfers between genetically distinct populations, which would result in a decrease in the genetic diversity of wild populations or the potential to introduce new sponge-associated microbe types into a region.\n
Simple
Identification info
- Date (Revision)
- 2022-11-21T00:00:00
- Website
- AIMS Web Site
- Website
- AIMS Web Site
- Credit
- Duckworth, Alan R, Dr (Principal Investigator)
- Status
- Completed
Principal investigator
Duckworth, Alan R, DrAustralian Institute of Marine Science (AIMS)
Point of contact
Data Manager, AIMS Data CentreAustralian Institute of Marine Science (AIMS)
- Temporal resolution
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P1Y0M0DT0H0M0S
- Topic category
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- Oceans
Extent
Extent
- Description
- Erub (Darnley Island)
Extent
Extent
- Description
- Ugar (Stephen Island)
Extent
Extent
- Description
- Poruma (Coconut Island)
Extent
Extent
- Description
- Warraber (Sue Island)
Extent
Extent
- Description
- Keats Island
Extent
Extent
- Description
- Kodall Island
Extent
Extent
- Description
- Collective resources start and end dates
Temporal extent
- Time position
- 2006-11-09
- Time position
- 2006-11-25
- Maintenance and update frequency
- As needed
Resource constraints
- Linkage
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http://i.creativecommons.org/l/by-nc/3.0/au/88x31.png
License Graphic
- Title
- Creative Commons Attribution-NonCommercial 3.0 Australia License
- Website
-
http://creativecommons.org/licenses/by-nc/3.0/au/
License Text
- Other constraints
- Use Limitation: All AIMS data, products and services are provided "as is" and AIMS does not warrant their fitness for a particular purpose or non-infringement. While AIMS has made every reasonable effort to ensure high quality of the data, products and services, to the extent permitted by law the data, products and services are provided without any warranties of any kind, either expressed or implied, including without limitation any implied warranties of title, merchantability, and fitness for a particular purpose or non-infringement. AIMS make no representation or warranty that the data, products and services are accurate, complete, reliable or current. To the extent permitted by law, AIMS exclude all liability to any person arising directly or indirectly from the use of the data, products and services.
- Other constraints
- Attribution: Format for citation of metadata sourced from Australian Institute of Marine Science (AIMS) in a list of reference is as follows: "Australian Institute of Marine Science (AIMS). (2013). Population genetics and microbial analysis of the Dictyoceratida sponge, Coscinoderma matthewsi, from central and eastern Torres Strait, Australia (MTSRF Project 1.3.2), https://apps.aims.gov.au/metadata/view/23f172bb-ac57-4b7f-abd4-044ca0b4daea, accessed[date-of-access]".
- Other constraints
- Resource Usage: \n Use of the AIMS data is for not-for-profit applications only. All other users shall seek permission for use by contacting AIMS. Acknowledgements as prescribed must be clearly set out in the user's formal communications or publications.\n
- Language
- English
- Character encoding
- UTF8
Content Information
- Content type
- Physical measurement
Distribution Information
Distributor
Distributor
AIMS Data CentreAustralian Institute of Marine Science (AIMS)
- OnLine resource
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Map
Map
- OnLine resource
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MTSRF Project 1.3.2
MTSRF Project 1.3.2
- OnLine resource
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Ecological role and potential value of sponges to Torres Strait: Duckworth AR, Wolff CWW, Cobb RE and Webster NS (2007) Ecological role and potential value of sponges to Torres Strait. Marine and Tropical Sciences Research Facility. 59 p.
Ecological role and potential value of sponges to Torres Strait: Duckworth AR, Wolff CWW, Cobb RE and Webster NS (2007) Ecological role and potential value of sponges to Torres Strait. Marine and Tropical Sciences Research Facility. 59 p.
Resource lineage
- Statement
- Statement: PCR reactions were performed as described by:Ferris MJ, Muyzer G and Ward DM (1996) Denaturing gradient gel electrophoresis profiles of 16S rRNA-defined populations inhabiting a hot spring microbial mat community. Appl Environ Microbiol 62: 340-346.with modifications to the reverse primer described in: Muyzer G, de Waal EC and Uitterlinden AG (1993) Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA. Appl Environ Microbiol 59: 695-700. \n
- Hierarchy level
- Dataset
- Maintenance and update frequency
- As needed
Metadata
- Metadata identifier
- urn:uuid/23f172bb-ac57-4b7f-abd4-044ca0b4daea
- Language
- English
- Character encoding
- UTF8
Type of resource
- Resource scope
- Dataset
- Metadata linkage
-
Point of truth URL of this metadata record
Point of truth URL of this metadata record
- Date info (Creation)
- 2013-02-28T00:00:00
- Date info (Revision)
- 2022-11-21T14:00:04
Metadata standard
- Title
- ISO 19115-3:2018