The effects of disease and stress on the microbial community of the sponge Ianthella basta

Sponges form a highly diverse and ecologically significant component of benthic communities. Despite their ecological importance, threats to populations and communities, including disease dynamics in sponges, remain relatively unexplored. There have been severe disease epidemics in sponges from the Caribbean and the Mediterranean; however, evidence of extensive sponge mortalities have not been reported in the Great Barrier Reef (GBR) or Torres Strait, northeastern Australia. This thesis describes a disease-like syndrome affecting the sponge Ianthella basta, a sponge that is common to both regions. The prevalence of disease as well as a comprehensive analysis of the microbial communities associated with affected and healthy sponges was determined. Manipulation of environmental parameters, including temperature and sedimentation were undertaken to establish potential causes of the disease-like syndrome. In addition, the microbial community associated with tissue regression of I. basta is also reported. Disease prevalence surveys were conducted in the Palm Islands on the central GBR and Masig Island, Torres Strait. Symptoms of the disease-like syndrome affecting I. basta included discolored, necrotic spots leading to tissue degradation, exposure of the skeletal fibers and disruption of the choanocyte chambers. Sponges were assigned to pre-determined disease categories using tissue necrosis and the presence of brown spot lesions as a proxy of health. Sponges with brown spot lesions were present at all sites with 43% and 66% of I. basta exhibiting disease-like symptoms in the Palm Islands and Torres Strait, respectively. Sponges from Torres Strait also showed a greater incidence of significant and extensive necrosis in comparison to sponges from Palm Island (11.5 vs. 6%). A comprehensive comparison of bacteria, viruses, fungi and other eukaryotes was performed in healthy and diseased I. basta using multiple techniques to ascertain the role of microbes in the disease process. A low diversity of microbes was observed in both healthy and diseased sponge communities, with all sponges dominated by an Alphaproteobacteria, a Gammaproteobacteria and a Thaumarchaea. Bacterial cultivation, community analysis by Denaturing Gradient Gel Electrophoresis (DGGE) (Bacteria and Eukarya), sequencing of 16S rRNA clone libraries (Bacteria and Archaea) and direct visual assessment by electron microscopy failed to reveal any putative pathogens. In addition, infection assays could not establish the syndrome in healthy sponges even after direct physical contact with affected tissue. These results suggest that microbes are not responsible for the formation of brown spot lesions and necrosis in I. basta. Elevated temperatures and other anthropogenic factors are increasingly being linked with disease in marine organisms. Manipulative experiments were undertaken to ascertain the potential role of environmental stressors (temperature and sedimentation) in the formation of brown spot lesions and necrosis in I. basta. In addition, the effects of elevated temperature, sedimentation and antibiotic exposure on the microbial community of I. basta were quantified. Neither elevated temperatures nor increased sedimentation induced the formation of brown spot lesions, indicating they are not responsible for the syndrome. However, sponges exposed to 32˚C developed substantial discoloration and deterioration of their tissues, resulting in death after eight days. The decline in sponge heath was accompanied by a shift in the microbial community, with higher diversity observed within the Alpha- and Gammaproteobacteria. Though this shift occurred, the microbial community was stable in sponges exposed to this temperature (32˚C) for the first four days of the experiment. No shifts in the microbial community of I. basta were observed with increased sedimentation and antibiotic exposure, with all dominant symbionts present (Alphaproteobacteria, Gammaproteobacteria and Thaumarchaea). Overall, these results indicate I. basta has a stable microbial community. The physiological stress response of tissue regression observed in heat-stressed I. basta was further explored. Following collection and transportation, tissues of I. basta often regress leaving visible subdermal gaps between primary fibers. Changes in the bacterial communities associated with each tissue state were also assessed using DGGE. Six necrotic specimens of I. basta and 12 healthy sponge explants were collected at Orpheus Is. , northeastern Australia and transported to aquarium facilities at the Australian Institute of Marine Science. After 12 h both the healthy explants and necrotic sponges displayed substantial tissue contraction with visible gaps evident between sponge skeletal fibers. However, within 72 h all sponges recovered to their original states. To quantify the level of tissue regression and subsequent recovery, a photo of each sponge was taken and images were compared using an integrated density measurement of Image Tool for Windows (UTHSCA). Histological samples were taken from sponge explants to compare cellular organization during this regression and recovery process. The integrated density of the sponge tissue effectively doubled within 72 h (increasing by 92%), confirming tissue recovery in I. basta. Histological analysis of the explants revealed that sponges affected by tissue regression had significantly fewer choanocyte chambers [(0.4 ± 0.1 vs. 4.5 ± 0.7) (mean ± S.E)] in regressed and recovered tissues respectively, and more densely packed granulated cells than recovered sponges. Principal Component Analysis of the DGGE data showed consistent patterns of microbial symbionts in both regressed and recovered sponges. This thesis represents a systematic approach to investigating disease in sponges, which can be used as a template for future research. Results obtained in this thesis contribute valuable baseline data on a disease-like syndrome affecting a common marine sponge and provide evidence that microorganisms are unlikely the causative agent. In addition, disease-like symptoms were never induced in response to temperature or sedimentation stress, indicating an environmental origin of the syndrome is also unlikely.