Complex interactions among amino acids, biofilms and settling larvae of the polychaete hydroides elegans

The polychaete Hydroides elegans is one of the major fouling organisms in Hong Kong and other tropical and subtropical waters. The process of larval settlement and metamorphosis of this species has been intensively investigated recently. Previous studies suggested that amino acids might be the natural inducers responsible for gregarious settlement of H. elegans on or near the conspecific adults and on the sympatric bryozoan Bugula neritina. My thesis research was focused on the complex interactions among amino acids, biofilms and settling larvae of the H. elegans from ecological and molecular levels. Twenty single amino acids and one mixture were tested in the larval settlement bioassays. Eleven amino acids directly induced the larval settlement in a concentration-dependent manner; isoleucine did not affect the settlement of the larvae; and the remaining eight amino acids and one inductive amino acid were toxic to the competent larvae at certain concentrations. In addition, the amino acid mixture made of aspartic acid and glutamic acid, showed a stronger inductive effect on larval settlement than single amino acids at the same concentrations. To investigate the indirect effect of amino acids on the larval settlement of H. elegans, two-day-old biofilms were first exposed to eight different amino acids or an amino acid mixture for 24 h, and then the bacterial abundance, bacterial community structure, and bioactivity of the treated biofilms were determined using DAPI staining, T-RFLP analysis and larval settlement bioassay, respectively. Aspartic acid, glutamic acid, and their mixture significantly increased bacterial abundance, modified the bacterial community structure on the biofilms, and elevated the inductive effect of the biofilms. Alanine and asparagine increased. Isoleucine decreased the bioactivities of the biofilms by changing the bacterial species composition of the biofilms. Distinct peaks in T-RFLP profiles were only found in the treated biofilms with enhanced/decreased inductive effect, indicating that the bacteria corresponding to these peaks might have been responsible for the change in the bioactivity of biofilms. Furthermore, the existence of glutamate receptors in H. elegans was confirmed using radio-ligand binding assays. By using homologic cloning, three fragments from genomic DNA of H. elegans and one fragment (glur-1) from cDNA were successfully obtained and sequenced. Blast analysis revealed that these fragments belong to the glutamate receptor gene family. The gene glur-1 was then used for semi-quantitative analysis of the gene expression level in different developmental stages of H. elegans. The results showed that the expression level of this gene reached the highest level when the larvae were metamorphosing, indicating that this gene played an important role in the larval settlement and metamorphosis of H. elegans