Investigation of genes involved in larval attachment and metamorphosis of biofouling species hydroides elegans and balanus amphitrite
by Honglei Li
Ph.D. Atmospheric, Marine and Coastal Environment
xx, 243 leaves : ill. (some col.) ; 30 cm
The polychaete Hydroides elegans and barnacle Balanus amphitrite are the major fouling organisms in Hong Kong and other tropical and subtropical waters. Structural and functional characterization of larval settlement related genes has contributed to a better understanding of how marine benthic larvae may respond to cues and proceed to attachment and metamorphosis....[ Read more ]
The polychaete Hydroides elegans and barnacle Balanus amphitrite are the major fouling organisms in Hong Kong and other tropical and subtropical waters. Structural and functional characterization of larval settlement related genes has contributed to a better understanding of how marine benthic larvae may respond to cues and proceed to attachment and metamorphosis.
Differential display was used to compare overall differences in gene expression between competent larvae and attached larvae of H. elegans. The competent larvae were triggered to attach by IBMX to yield the synchronal settlement, three differentially expressed DNA fragments were isolated encoding BTB/POZ domain protein, ATP synthase F0 C subunit, and a hypothetical protein. Three cDNA libraries were constructed from H. elegans competent larvae, attached larvae, and adults RNA. In our study, 83 cDNA clones were sequenced in attached larvae library. Four functional genes encoding ATP synthase F1 epsilon subunit, Ran GTPase binding protein, NADH dehydrease, and cell division cycle protein were isolated. In addition, the catalytic (PKAc) and regulatory (PKAr) subunits of cAMP-dependent protein kinase A (PKA) genes from H. elegans were cloned, and their temporal and spatial expressions in different developmental stages of H. elegans were examined using quantitative real-time PCR and in-situ hybridization. Our results indicated that the PKA genes may play important roles in mediating larval development of H. elegans.
Furthermore, the above nine genes were selected and the primers were designed for real-time PCR analysis using RNA extracted from larvae treated with different inducer and antifouling compounds. Antifouling compound treatments such as genistein, branched fatty acid, and butenolide exerted different effects on the expression of each gene showing the different levels of regulation happened during the larval settlement process. Signal transducer genes (PKA genes) and two regulatory genes (BTB/POZ domain protein gene, Ran-GTPase binding protein gene) were proved to be good marker genes to monitor the larvae-chemical interaction.
To investigate the influence of settlement cues (i.e. natural biofilm and conspecific settlement factor), antifouling compounds (i.e. genistein and butenolide), and 0.22 μm filtered sea water (control) on the expression profile of barnacle cypris specific genes (bcs genes) in B. amphitrite, quantitative real-time PCR was used to better understand the mechanism of larval attachment and metamorphosis. Exposure of cyprids to environmental cues did not enhance the down-regulation of bcs-1 to -5 in cyprids in order to increase the rate of attachment, however, the expression of bcs-6 increased significantly before the commencement of attachment. Antifouling compound genistein up-regulated the bcs-1 expression and inhibit bcs-6 expression, however, butenolide decreased tested bcs-1, -2 and -6 gene expression levels. Our results thus confirmed that bcs-6 is involved in larval attachment and metamorphosis. Our findings here, which describe the relationship between environmental cues, antifouling compound treatments, and the expression of bcs genes during cypris metamorphosis lay a solid foundation for future studies on the molecular mechanism of larval settlement in barnacle species.