THE GROWING COMPLEXITY OF BARNACLE CYPRID GLUE  

Spectral observations from our prior collaboration (Nature Communications 5:4414) revealed that the permanent adhesive plaque of the cyprid contained not only phosphoproteins but lipids as well.  Although this was a significant finding, there were still more unidentified peaks and stretching regions denoted in the broadband CARS imaging spectra. Furthermore, the Pacific oyster genome also revealed an increase in the expression of both chitin synthase and a fibronection-like protein at the time of pediveliger larval attachment to the substratum. Based on these observations and the fact that cyprids produce a cuticle of chitin, we expressed a chitin binding domain protein (CBD) that had a known binding domain for the insoluble form of the polysaccharide.  The CBD plasmid was expressed and harvested in our lab from bacterial cells, purified by a nickel column and labeled with Alexafluor 546 for fluorescence.  To test for function and to validate the molecular probe we dissected the midgut from mealworms and used the probe to detect the presence of the peritrophic membrane. After successful validation of the CBD probe specificity, 3 day old cyprids from the Striped barnacle, (Amphibalanus amphitrite)  at 18 hours post attachment and adherent oyster hemocytes  from the Eastern oyster (Crassotrea virginica) were tested and imaged on a Leica SPE DMI4000 LSM.

Results (shown here) indicate that chitin is present in the permanent adhesive disc of attached cyprids. We also imaged adherent oyster hemocyte aggregations which also indicated the presence of chitin in these cells.  Prior peer reviewed research on squid has shown that molluscan hemocytes do contain chitin so we treated this result as a positive control. These findings imply a far greater complexity to the formation of the adhesive then what was previously realized.