Penaeid cell culture: Characterization of cytoskeletal components and detection of cell cycle regulatory elements ((cdc2)p34 and cyclins A and B)

Dwaine Alan Braasch


Considerable effort has been put into the development of crustacean cell culture during the past forty years. Many of these studies have left researchers frustrated and puzzled, but none have produced a cell line that can be sustained in vitro . This investigation is an extension of previous efforts in penaeid cell culture and focuses on advanced methodologies. A total of 89 combinations of basal cell culture media and various additives such as serum, growth factors, attachment factors, trace minerals, and mutagens were evaluated for their ability to stimulate cells and tissues from penaeid shrimp. Cultures were maintained for up to four weeks at room temperature in the absence of light. No passageable cell lines were produced from these efforts and the majority of cultures succumbed to bacterial or fungal contamination. In response to the failed attempts to culture cells from penaeid shrimp, the mitotic state of hemocytes was determined by immunostaining cytoskeletal components, including actin, myosin, α-tubulin, β-tubulin, vimentin and clathrin. The immunostaining data established the presence of structural components such as actin and myosin, and the lack of structures associated with mitosis such as mitotic spindles. These data support the conclusion that hemocytes are terminal populations of cells. Studies were conducted to characterize cellular components governing cell division. The main objective of this portion of experimentation was to confirm the presence or absence of the cell cycle regulatory proteins cyclin A, cyclin B and p34cdc2 in proteins extracted from egg and muscle tissues from Penaeus vannamei . Western blot analysis using anti-S. solidissima cyclin A and cyclin B antibodies were capable of detecting cyclin homologues from protein extract separated on denaturing polyacrylamide gels. The molecular weights of cyclin A and cyclin B from P. vannamei were determined to be 45 and 54 kD, respectively. The MPF (mitosis promoting factor) cofactor p34cdcd2 was also detected by western blot analysis of the same protein extracts with an anti-PSTAIRE antibody.