Using next generation sequencing methods to understand biofouling assemblages

Man-made infrastructure has now become a common feature of the coastal environments due to increasing human activities. However, the introduction of these structures has caused both ecological and environmental impacts. They have also been considered the first point of entry for introduced species that are brought by shipping. The study of biofouling assemblages that develop on these hard surfaces showed that introduced species were more likely to settle on these artificial structures than native species. The development of biofouling assemblages is usually influenced by both biotic and abiotic factors and the study of biofouling assemblages over time will provide an insight into the effects of anthropogenic activities on the growth of biofouling organisms. This was achieved by observing and assessing the development of biofouling assemblages at several time points in places with different levels of human activities. Biodiversity assessment has been carried out in many ecological studies using metabarcoding in recent years and settlement plates have been widely used for studies on biofouling. The main aim of this thesis was to examine the efficacy of metabarcoding in the early detection of introduced species and to assess the development of biofouling community in Gippsland Lakes, Victoria and Gulf of St Vincent, South Australia over a period of six and twelve months, respectively.<br><br>To study biofouling assemblages, Perspex settlement plates were deployed at several sampling sites in Gippsland Lakes, Victoria and Gulf of St Vincent, South Australia for up to six and twelve months, respectively. Plates were removed at one, three and six months of deployment in Gippsland Lakes and at one, three, six, nine and twelve months of deployment in South Australia. The biofouling material collected was processed for DNA extraction. Over time, the biofouling assemblages increase, and to provide an accurate biodiversity assessment of the biofouling assemblage, a large amount of biofouling material (>10 g) is required. However, standard DNA extraction kits (PowerMax® Soil DNA Isolation Kit) only allows up to 10 g of sample to be processed at a time. I assessed the suitability of freeze drying to process biofouling samples that weigh more than 10 g in Chapter 2. This method allowed removal of water from samples, reducing their weight and allowing them to be efficiently processed for DNA extraction. Importantly, the purity of the extracted DNA was not compromised, allowing it to be used as a template for PCR using the primers which amplify the hypervariable V4 region of the 18S ribosomal RNA. The amplicons were further used for high throughput sequencing for all chapters looking at development of biofouling assemblages. This study showed that the use of freeze drying is an effective tool for processing large mass of samples including biofouling samples for the purpose of studying biodiversity.<br><br>In Chapter 3, I discuss the use of settlement plates and metabarcoding as a potential non-invasive approach for early detection of introduced species. In order to detect the presence of introduced species at a stage where the organisms were not readily identifiable, i.e. at their early life history stages, biofouling samples collected from Gippsland Lakes after one month of plate deployment were assessed. My findings showed that commonly known marine invasive species (MIS) such as Asian bag mussel Arcuatula senhousia were detected. A quantitative polymerase chain reaction (qPCR) analysis confirmed its detection. The introduced sea grape tunicate Molgula manhattensis was detected and its last reported detection in Australia was in 1976. Further molecular work using PCR-based Sanger sequencing of 28S marker and BLAST analysis confirmed the identity. Furthermore, the findings from this study showed that introduced species can be detected using settlement plates and metabarcoding when the organisms were at their early life history stages on the settlement plates.<br><br>The ability of metabarcoding to produce a large database of DNA sequences to identify organisms makes it a robust tool for assessing biodiversity. The combined use of metabarcoding and settlement plates provided baseline information on the biodiversity in biofouling assemblages in the Gippsland Lakes, Victoria (Chapter 4) and South Australia (Chapter 5). In an estuarine system such as the Gippsland Lakes, the differences in species composition of the biofouling assemblages were attributed to the location of the sampling sites. Moreover, differences in salinity could account for the different species assemblages at the three locations. The difference in recreational boating traffic is also a possible factor in influencing the species composition in Paynesville and Metung. In order to assess the differences in biofouling assemblages in places of varying anthropogenic activities, I have also examined the species composition of biofouling assemblages in several locations in South Australia for a span of twelve months. To the best of my knowledge, this is the first study that utilises metabarcoding to study development of biofouling assemblages over a period of twelve months. The findings from this chapter show that the species composition among all locations differed. The highest difference was observed between Kingscote and North Haven and the presence of introduced species had the highest effect in North Haven, which is considered to be a highly disturbed area. For example, the ascidian Ciona intestinalis dominated the biofouling community in North Haven over twelve months while ascidian Ascidiella aspersa was most dominant in the first six months in North Haven. Frequent shipping among North Haven, Wirrina Cove and Christmas Cove may have encouraged transfer of species as the species composition in the biofouling communities had high level of similarity. However, Wirrina Cove and Christmas Cove had higher similarity which could be attributed to the similarity in the environmental conditions. Findings from this chapter provide an insight into understanding how environmental factors and human activity affect the growth of biofouling assemblages.<br><br>In summary, the findings from this thesis provided baseline information on the biofouling assemblages found on the settlement plates in Gippsland Lakes and Gulf of St Vincent. This tool is a non-invasive approach which allowed the detection of known and new introduced species. My research highlighted the changes in the successional stages of biofouling assemblages in places with different levels of anthropogenic activities. Moreover, the findings in this thesis showed that the presence of introduced species, especially in areas of high anthropogenic activity, may affect the biofouling assemblages. This study highlights the importance of understanding the ecological impacts of man-made structures in the environment and the need to carry out regular monitoring especially in smaller marinas and domestic ports.