Determining the Diet of Spiny Lobster Phyllosomas: Using a Prey Enriched, Amplicon Pyrosequencing Approach

Discovering the natural diet of spiny lobster larvae (phyllosomas) could give insight into how an aquaculture feed could improve their survival. It could also lead to improving the sustainable management of fisheries stocks by enabling us to understand how prey dynamics affect the subsequent recruitment of lobster post-larvae into fisheries. However, the natural diet of phyllosomas has been difficult to determine because of the low densities, remote offshore habitat, and flat transparent morphology of the larvae that makes them difficult to dissect and because their gut contents contain no morphological clue of their prey. A DNA approach overcomes these problems, but creates alternative problems: phyllosomas are generalist predators, making it difficult to detect prey without co-detecting lobster DNA, guts contain additional ???noise??? such as fungi, and net sampling increases risk of exogenous contamination being detected by PCR. The present thesis addressed these problems and successfully developed a novel approach to extract uncontaminated gut contents using inexpensive high-grade syringes, and then applying a DNA-based protocol for extracting and determining the diet of phyllosomas. This protocol targeted small hyper-variable regions of the 18S rDNA gene and a lobster-specific peptide nucleic acid clamp (PNA-clamp) suppressed predator PCR amplification. To overcome the potential ???noise??? that comes from amplifying gut contents, this study generates an excess of reads by high throughput 454 pyrosequencing. This prey-enriched, amplicon pyrosequencing protocol was used to determine the prey of phyllosomas of two species of spiny lobster: Jasus edwardsii and Panulirus cygnus. A DNA approach had not previously been applied to these species. It was found that both species have a taxonomically diverse diet, but that some diet items such as hydrozoans and bony fishes are especially significant. Additionally, colonial radiolarians are significant diet items for P. Cygnus, as are ctenophores for J. edwardsii. Other significant, although less abundant, diet items for both species are salps, arrow worms and krill. Overall, these results indicate that while phyllosomas are generalist zooplankton predators they also have preferences for some prey taxa. Significantly, the prey-enriched pyrosequencing approach that was developed in this thesis revealed that there are no significant dietary differences between P. cygnus phyllosomas in the East Indian Ocean across distinct water masses. This is despite the fact that phyllosomas sampled from distinct water masses are in varying nutritional condition, and dietary composition had previously been hypothesised as contributing to phyllosoma condition.