Bacterial challenge and eicosanoids act in plasmatocyte spreading

We report on experiments designed to more thoroughly document the roles of eicosanoids as crucial elements in cell spreading and on experiments designed to test the hypothesis that in vivo bacterial infections influence cell spreading on glass surfaces. We used hemocytes prepared from tobacco hornworms, Manduca sexta (L.) (Lepidoptera: Sphingidae) and four species of bacteria ( Serratia marcescens , Escherichia coli , Bacillus subtilis , and Micrococcus luteus ) in each experiment. Our protocols yielded several important points: (i) hemocytes prepared from hornworms at 15 and 60 min following infection with, separately, each of the four bacterial species were fundamentally altered in size (all less than the 15μ m counting cut-off) and none of the hemocytes exhibited cell-spreading behavior; (ii) the influence of bacterial challenge on cell spreading declined with incubation time post-challenge; (iii) conditioned medium (CM) prepared by exposing hemocytes to bacterial cells in vitro exerted a strong dose-dependent influence on cell spreading. Specifically, plasmatocytes increased in length from about 38 μ m with 2.5% CM to a maximum of about 54 μ m at 100% CM; and (iv) the retarding influence of dexamethasone (an eicosanoid biosynthesis inhibitor) on cell spreading was reversed by arachidonic acid, prostaglandin H 2 , and CM. Taken together, these findings indicate that both bacterial infection and eicosanoids influence hemocyte-spreading processes.