Gill lateral cells (GLC) of Crassostrea virginica are innervated by serotonin and dopamine nerves from their ganglia. Most bivalves have GLC cilia that respond to serotonin and dopamine, with serotonin being the neurotransmitter that increases GLC cilia beating rates and dopamine being the neurotransmitter that decreases beating rates. The motor aspects of GLC innervation have been well studied, but there is limited information about sensory inputs. Our previous work found C. virginica senses light and other cues, such as food and crab extract when applied to the mantle rim and adjusts GLC cilia beating rates accordingly. Shining light on the mantle rim decreased GLC cilia beating rates, but when shined directly on the gill had no effects on cilia beating rates. This light-sensitive reaction was duplicated by applying histamine to the mantle rim and blocked by applying the histamine antagonist, famotidine, prior to shining light. Recently, published articles have found the presence of a rhodopsin-l ike g ene in a dult Crassostrea gigas . We hypothesize that in C. virginica the p hotoreceptor, rhodopsin, is present in mantle rim tentacles. To test this, we used immunohistofluorescence (IHF) microscopy and Western Blotting (WB) on mantle rim tentacles to view and detect the presence of rhodopsin. Mantle rims were excised from adult C. virginica a nd prepared for IHF or for PAGE followed by WB. Briefly, for IHF, mantle rims sections were snap frozen, cryostat sectioned, fixed with EDAC (N-Ethyl-N’-(3-dimethyl-aminopropyl) carbodiimide hydrochloride), treated with blockers, and incubated with rhodopsin primary antibodies and FITC secondary antibodies. Sections were viewed on a Leica epilume fluorescence microscope with a Leica DFC400 camera, 50 watt mercury lamps and FITC excitation/emission filters. For WB, mantle rims sections were homogenized in detergent and centrifuged. Protein concentrations of the supernatant were determined by detergent compatible Bradford. Aliquots were treated with Laemmli followed by PAGE (20-40 µg protein/well). After electrophoresis gels were sandwiched for transfer onto nitrocellulose membranes. After WB membranes were blocked, then incubated with rhodopsin primary antibodies and HRP secondary antibodies in TBST and 2% blocker at 4°C. Blots were resolved by KPL TrueBlue Peroxidase Substrate and viewed. IHF showed the presence of rhodopsin in mantle rim tentacles. WB detected discrete bands of rhodopsin protein present in mantle rim tentacles as well. The study shows the presence of the light sensitive receptor rhodopsin in mantle rim tentacles of adult C. virginica and complements other work of our lab that showed a neurophysiological response to light when shined onto the mantle rim. It also continues to demonstrate that C. virginica is a useful model to study neurophysiology as well as the pharmacology of drugs affecting the nervous systems.
This work was supported in part by grants 2R25GM06003 of the Bridge Program of NIGMS, 0537231071 of the CSTEP Program of NYSED, P120A210054 of the MSEIP Program of the DoEd, and NIH grant K12GM093854-07A1 IRACDA Program of Rutgers University.