Thesis

Development of an enzyme-linked immunosorbant assay to determine hemolymph levels of vitellogenin in Petrolisthes cinctipes and Petrolisthes manimaculis

Petrolisthes cinctipes and P. manimaculis are two closely related species of anomurans that live in the upper intertidal zone along the Central California coast. Morphometric measurements were collected to gain insight on the reproductive cycles of these species. Ovary weights of P. cinctipes increased from July (0.2 ? 0.1 ?g) to September (18.0 ? 6.0 ?g), remained similar through January (19.0 ? 7.0 ?g), and then decreased in February (5.6 ? 3.0 ?g). Vitellin (Vn), an egg yolk protein, is metabolized from a larger hemolymph protein, vitellogenin (Vg). The Vns of both species were isolated and characterized. Using SDS-PAGE it was determined that the Vn of the conspecifics, P. cinctipes and P. manimaculis, consist of three major subunits that have a MW of 93 ? 2 kDa, 82 ? 2 kDa, and 65.7 ? 1.4 kDa. Two minor bands were also detected at 111 ? 2.3 kDa and 40 ? 1.3 kDa. Using HPLC, the native molecular mass of P. cinctipes vitellin was found to be 301 ? 14 kDa with a small doublet. The native molecular mass for P. manimaculis Vn is 324 ? 11 kDa with a more pronounced doublet of 160 ? 13 kDa. A Western blot was used to test the reactivity of the Petrolisthes Vn with various antibodies. It was revealed that two of the major Petrolisthes Vn subunits, 93 ? 2 kDa and 65.7 ? 1.4 kDa, successfully bound with Homarus anti-Vn antibodies. An ELISA was developed that can measure Vn and Vg in the hemolymph of both Petrolisthes species, with an effective range from 9-3000 ng. There was no difference in mean Vg level from September (240 ? 100 ?g/mL) through January (310 ? 50 ?g/mL), with a total mean Vg concentration of 284 ? 32 ug/mL.

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