Influence of size and spatial competition on the bioactivity of coral reef sponges from Torres Strait (MTSRF Project)

Size frequency surveys of the sponges Coscinoderma matthewsi, Hyrtios erecta and Ianthella basta (yellow color morph) were conducted at Masig Island, central Torres Strait, in March 2007. At each of eight sites, separated by at least 200 m, three randomly positioned 30 x 1 m transects were surveyed, with each transect separated by at least 20 m. All transects were located on sloping reef at between 10 and 12 m depth.The greatest dimension of each sponge was measured for Coscinoderma matthewsi and Ianthella basta, and used as an approximation of overall sponge size. Due to the branching morphology of Hyrtios erecta, the length of each individual branch was included to give an overall measurement. Sponges were also assigned a competition class based on the percentage of their perimeter in contact or within 1 cm of surrounding or encroaching organisms.Three tissue samples, each 1-2 cm³ were collected from 20 different individuals of each species from a single site to examine the influence of sponge size on bioactivity. Prior to sampling, sponges were also measured for size, photographed and assigned a competition class. The sponges sampled encompassed the full size range of each respective species, except for very small sponges, which did have sufficient tissue to obtain three independent tissue samples. Sponges displaying signs of disease were not sampled. Samples were frozen at -20°C within one hour of collection and kept frozen until freeze dried.Freeze-dried sponge tissue samples were weighed and extracted 3 times with methanol (MeOH). Crude extracts were made up into a 100 mg/ml concentration in MeOH based on individual extract weights. 100 µl of each sample was then aliquoted to microtitre plates and dried down, yielding 10 mg of dry extract per sample for bioactivity screening.The production of bioactive metabolites and overall bioactivity of sponges was examined using the sulforhodamine B (SRB) assay. Crude extracts were re-suspended in dimethyl sulfoxide (DMSO) and added to 96 well plates containing 100 µl of pre-incubated cell suspension. Several concentrations were tested for each species, with the concentration showing the greatest fine-scale variation in toxicity between individual samples selected for the analyses: 100 µg/ml for Coscinoderma matthewsi; 0.1875 µg/ml for Hyrtios erecta; and 100 µg/ml for Ianthella basta. One replicate plate was made for each sample plate and for each concentration, with appropriate controls. After 48 hours of incubation, plates were fixed with trichloroacetic acid and stained using 0.4% SRB. Plates were read using the Wallac Victor plate reader at an absorbance of 490 nm, and % cell growth values were calculated.