In Australia the tropical abalone, Haliotis asinina Linnaeus, has recently received attention for its potential as an aquaculture species and as a model organism for improving cultivation methods of abalone and other gastropods. In this thesis I combine a number of 'whole organism' experiments with various molecular techniques to investigate recognised production bottlenecks in abalone aquaculture, with a major emphasis on settlement and metamorphosis.
I initially describe a set of techniques for the efficient production of competent H. asinina larvae. These include practices for the transport and maintenance of broodstock, spawning and fertilisation, rearing of larvae to competency, and induction of settlement and
metamorphosis. I also identify a formulated feed that promotes high juvenile growth. Based on my analyses, the optimal temperature range at which to rear H. asinina larvae is 25°C to 28°C. Through a series of settlement assays, I determined that a non-geniculate coralline alga (NCA), tentatively identified as Mastophora pacifica, is a potent inducer of H. asinina settlement and metamorphosis. In contrast, exposure of competent larvae to a wide range of GABA and KCI concentrations did not induce metamorphosis. Assessment of the suitability of four artificial diets (formulated for temperate Australian abalone species) for grow out of H. asinina revealed two that promoted growth rates equivalent to those produced by a natural macroalgal diet.
I then determined the optimal age at which to induce larvae to settle and metamorphose with the NCA treatment. The results of these experiments revealed that the majority of H. asinina larvae are competent to metamorphose at 84 to 90 hours after fertilisation, but that there is considerable variation in the age at which individual larvae attain competency. I also conclude that the development of the radula can be used as an indicator of competence. A subsequent experiment demonstrated that a smaller proportion of younger larvae will metamorphose for equivalent NCA exposure times in comparison to older larvae. These experiments also demonstated that larvae become habituated to the NCA cue if exposed to it prior to attaining competency.
Having identified the age at which H. asinina larvae become competent to settle and metamorphose, I isolated a number of ESTs with temporal expression profiles that suggest they may be involved in the development of the competent state using a differential display technique. Spatial characterisation of these cDNAs by in situ hybridisation revealed a number of interesting expression patterns. One gene (designated B9), which was temporally expressed in pre-competent and competent veligers, was spatially restricted to the cephalic tentacles and propodium of the foot in competent veligers, suggesting that it may play a chemosensory role. Another gene (B15), which was only expressed in competent veligers, was spatially restricted to the mantle tissue, implying it plays a role in biomineralisation. Four other genes (B4, B13a, B13b and B13c), with significantly
different temporal expression profiles, displayed similar patterns of spatial expression being restricted to a set of 6 cells in the posterior region of the developing gut; it is unclear if these patterns overlap. One of these genes (B13a) showed significant sequence similarity to the ribosomal protein L28.
In the final data chapter I describe the construction of a λ cDNA library from a range of developmental stages and its partial characterisation by the sequencing of 234 clones. Searching the NCBI databases with these sequences revealed a number of proteins previously not recorded from gastropods, and in some cases lophotrochozoans. Having validated this library I used it to construct a novel microarray DNA chip. As a validation of this chip and probe labelling methods, I hybridised unfertilised egg
and competent veliger RNA onto this chip, and sequenced those clones that were up-regulated in competent veligers. Differential expression of these clones by RT-PCR confirmed the microarray profiles. I then went on to conduct hybridisations between competent veligers and recently metamorphosed postlarvae in order to infer global changes in gene expression during early metamorphosis. Significant differences in the expression of a number of housekeeping genes during metamorphosis between Drosoptiila and H. asinina were detected.
By identifying genes involved in the attainment of competence and settlement and metamorphosis, we can begin to dissect the molecular mechanisms that regulate these processes. This in turn may allow rates of settlement and metamorphosis of abalone and
other gastropods to be manipulated by a variety of techniques including genetic transformation and artificial induction of settlement and metamorphosis. These techniques should become viable alternatives to the traditional culture methods that rarely realise settlement rates greater than 20%.