The barnacle Balanus (=Amphibalanus) amphitrite is one of the globally distributed and a major component of rocky shore intertidal communities. The transition from its swimming larvae to sessile adults is referred as larval settlement, which has been a major target in studies of intertidal ecology and antifouling strategies. However, the lack of genomic information hinders a comprehesive understanding of the detailed molecular mechanism of larval settlement. Therefore, the main goal of my thesis work was to profile and characterize the genes possibly involved in larval settlement of B. amphitrite by using pyrosequencing, shotgun proteomics technology, and molecular biology approaches.

First, the transcriptome of B. amphitrite was sequenced by using 454 pyrosequencing. In total,...[ Read more ]

The barnacle Balanus (=Amphibalanus) amphitrite is one of the globally distributed and a major component of rocky shore intertidal communities. The transition from its swimming larvae to sessile adults is referred as larval settlement, which has been a major target in studies of intertidal ecology and antifouling strategies. However, the lack of genomic information hinders a comprehesive understanding of the detailed molecular mechanism of larval settlement. Therefore, the main goal of my thesis work was to profile and characterize the genes possibly involved in larval settlement of B. amphitrite by using pyrosequencing, shotgun proteomics technology, and molecular biology approaches.

First, the transcriptome of B. amphitrite was sequenced by using 454 pyrosequencing. In total, 630,845 reads were obtained from the larval and adult stages. Among 92,322 predicted open reading frames, the annotations of 31,720 matched hits in the NCBI NR database. Among 7,954 putative genes differentially expressed between the two stages, I found that 1) vitellogenin that was uniquely expressed in the late nauplius stage may be an energy source for the subsequent non-feeding cyprid stages; 2) 20-kDa cement protein homologues that were expressed in cyprid cement glands probably function during cyprid attachment; and 3) receptor tyrosine kinases that were highly expressed in the cyprid stage may be involved in signal perception during larval settlement.

Second, the transcriptome sequenced allows high throughput gel-free proteomics analysis at different developmental stages of this species, including stage II nauplii, stage VI nauplii, cyprids and juveniles. More than 1,800 proteins were obtained from each developmental stage; 64 of which were identified as the differentially expressed proteins in the cyprid and juvenile stages. For instance, neuroglian cell adhesion molecule and ferrin that were up-regulated in the cyprid stage may play important roles in larval settlement. Besides, proteins that are participating in extracellular matrix, transcription and translation, programmed cell death, and cell differentiation were drastically up-regulated in juveniles, indicating their crucial functions in tissue remodeling and development in larval metamorphosis.

Third, detailed characterization was performed on selected genes of interest. Specifically, lectin genes containing two C-type lectin domains were cloned from B. amphitrite. They were highly expressed in late nauplius stage and were localized in the labrum part of nauplius where numerous chemoreceptors locate. Furthermore, the gene expression as dot distribution on cyprid carapace coincided with the localization of the putative sensory organs. Similarly, calmodulin gene (CaM) with conserved Ca²⁺-binding motifs was cloned; CaM was up-regulated in the cyprid stage and highly expressed in such tissues as compound eyes, posterior ganglion and cement glands that have essential functions in barnacle larval settlement. Bioassay shows the possible involvement of CaM and CaM-dependent myosin light chain kinase (MLCK) during barnacle larval settlement. In addition, another CaM gene from the subtidal polychaete Hydroides elegans was cloned and characterized. Our findings here profiled the transcritpome and proteome of B. amphitrite, and characterized selected genes in larval settlement of B. amphitrite and H. elagans, therefore encourage further exploration of larval settlement pathways in these two important marine species.