Surface-based technologies and the settlement of Mytilus galloprovincialis

The mussel Mytilus galloprovincialis is a common aquaculture species, and also a major fouling organism that has negative economic impacts. There are no standard assay conditions for this important species and therefore the initial study of this thesis (Chapter 2) quantified the effect of key factors on the settlement of pediveligers and plantigrades to provide a standardised and reproducible assay method. Density dependent settlement did not occur for either pediveligers or plantigrades. Settlement increased in drop assays in a 12 h light:12 h dark cycle, while bottom shade had no effect of any magnitude. In addition, settlement was significantly enhanced by storing pediveligers for between 4 and 24 days at 4oC. Overall, these data provide the template to optimise and standardise static laboratory settlement assays for mussels in order to develop materials that either enhance settlement for the aquaculture industry, or deter settlement for antifouling applications. Furthermore, simple mechanisms such as storage at 4oC can enhance settlement beyond current methods used in aquaculture hatcheries. In the third chapter of this thesis, key properties affecting the settlement and adhesion of marine invertebrate larvae were investigated. Surface wettability and microtopography can either enhance or deter larval settlement of many sessile marine organisms. To determine the effect of these surface properties on the settlement of pediveligers of M. galloprovincialis (Chapter 3), polymers spanning a range of wettability and microtextured polydimethylsiloxane (PDMS) were used. Furthermore, the adhesion strength of settled pediveligers on microtextured PDMS surfaces was quantified using a flow chamber. Settlement was enhanced at the hydrophilic end of the wettability spectrum, where mean settlement on nylon reached 33.5 ± 13.1%. In contrast, mean settlement on the most hydrophobic polymer, PDMS, was 4.2 ± 3.2%. Microtopography had a much stronger effect than wettability, where 400 μm textured PDMS enhanced settlement above 90%. Settlement preferences were also positively correlated to adhesion strength at flow rates of 4 knots, with all initially settled pediveligers on smooth PDMS detaching, while 79.9 ± 5.7 % of pediveligers remained on PDMS with a 400 μm texture. This process was further developed in the subsequent chapter (Chapter 4). The global mussel aquaculture industry uses specialised spat catching and nursery culture ropes made of multifilamentous synthetic and natural fibres to optimise settlement and retention of mussels on ropes for on-growing. However, the settlement ecology and preferences of mussels are poorly understood and only sparse information exists in a commercial context. This study quantified the settlement preferences of pediveligers and plantigrades of M. galloprovincialis for increasingly complex surfaces and site-specific settlement locations on and within ropes at an industrial scale using optical microscopy and X-ray micro-computed tomography (μCT). M. galloprovincialis has clear settlement preferences for increasingly complex materials and high selectivity of settlement sites relative to the size of individuals from the pediveliger stage through to the plantigrade stage. Pediveligers of M. galloprovincialis initially settle inside ropes and move outwards as they increase in size. In contrast, smaller individuals that have not grown move deeper inside of the ropes over time. This study demonstrated that μCT is an excellent non-destructive technique for mapping attachment sites of individuals as early as 1 day post settlement. Furthermore it quantified the numbers of settled individuals on and within ropes providing for the development of technologies to optimise aquaculture practices. Finally, in the last chapter of this thesis (Chapter 5), surface technologies to deter the settlement of pediveligers and plantigrades of M. galloprovincialis as fouling organism were investigated. Fouling-release materials minimise the adhesion of biofouling and are currently used as an antifouling strategy with non-target effects. Technologies to control the settlement and adhesion of the important fouling organism M. galloprovincialis on fouling-release materials were developed by incorporating the nanofillers titanium dioxide (TiO₂) and carbon nanotubes (CNTs) in PDMS matrices. The incorporation of TiO₂ prevented larval settlement when photoactivated with UV light, even at the lowest concentration of the nanofiller (3.75 wt%). Notably, there was 100% mortality of pediveligers exposed to photoactivated TiO₂. However, plantigrades initially settled to photoactivated TiO₂, but their adhesion strength was significantly reduced on these surfaces in comparison to blank PDMS. In addition, plantigrades had high mortality after 6 h. In contrast to the enhanced antifouling and fouling-release properties of PDMS filled with TiO₂, the incorporation of CNTs into PDMS had no effect on the settlement and adhesion of M. galloprovincialis. In conclusion, this study provides an optimised and standardised static laboratory settlement assay for mussels. Subsequently, surface-based technologies to control the settlement of M. galloprovincialis were investigated to develop materials that either enhance settlement and retention for the aquaculture industry, or prevent settlement thereby reducing biofouling. Microtopography and complexity of surfaces are both settlement cues for pediveligers, with complexity playing a key role for settlement and selection of attachment sites. These results highlight the efficacy of multi-filament ropes used in the mussel aquaculture industry as these complex materials allow pediveligers to settle at protected sites within the ropes and migrate to the exterior of the rope post-metamorphosis as individuals increase in size. They also provide a new and fundamental understanding of the settlement process to optimise the retention of hatchery reared mussel larvae to support a sustainable mussel aquaculture industry. Finally, in an attempt to reduce the negative economic impacts of mussels as a fouling organism by improving antifouling and fouling-release properties against M. galloprovincialis, TiO₂ was incorporated into coatings and was highly effective in preventing larval settlement, causing high rates of mortality and weakening the adhesion strength of any settled organism. In contrast, the incorporation of CNTs as nanofiller was ineffective. While TiO₂ has limitation in its application, it can provide an alternative non-biocidal antifouling strategy in the photic zone. In summary, the outcomes of this thesis provide a novel and innovative contribution to understanding the mechanisms driving mussel settlement preferences and technologies to control mussel settlement and growth.