Antagonistic effect of juvenile hormone on hemocyte-spreading behavior of Spodoptera exigua in response to an insect cytokine and its putative membrane action.

Juvenile hormone (JH) acts on membrane of follicle cells to induce ovarian patency for vitellogenesis, though it regulates various other physiological processes via putative intracellular receptors. This study suggests another JH membrane action by analyzing in vitro hemocyte behavior. In response to nonself, both granular cells and plasmatocytes of Spodoptera exigua can exhibit cell shape changes through spreading behaviors. Plasmatocytes were separated from total S. exigua hemocytes by Percoll gradient and exposed in vitro to an insect cytokine, plasmatocyte-spreading peptide (PSP), identified from Pseudoplusia includens. In response, the purified plasmatocytes spread in a dose-dependent manner from picomolar to micromolar concentrations. Interestingly, the PSP responses of plasmatocytes in S. exigua varied among different larval ages during fifth instar ( approximately 5 days at 25 degrees C) in a sensitivity order of late (5 days old)<early (1 day old)<mid (3 days old). Considering the overall endocrine changes that occur during the final instar of holometabolous insects, we suspected that JH and ecdysteroid hormones were responsive for this developmental modulation of plasmatocyte sensitivity to PSP. We tested this hypothesis by exposing plasmatocytes to hormone agonists in vitro. Pyriproxyfen, a JH agonist, significantly inhibited plasmatocyte sensitivity to PSP. JH I and II had significant effects on antagonizing plasmatocyte sensitivity to PSP, but either JH III or farnesoic acid did not. In contrast, 20-hydroxyecdysone (20E) enhanced the plasmatocyte sensitivity to PSP. Ethoxyzolamide, a putative JH competitor to membrane receptor, inhibited JH action on the plasmatocyte sensitivity to PSP. Though staurosporine (a protein kinase inhibitor) alone did not influence plasmatocyte sensitivity to PSP, it antagonized the JH inhibitory effect on the plasmatocytes. Ouabain, a specific Na+ -K+ ATPase inhibitor, also masked the JH action on the plasmatocytes. These results suggest that the JH acts on the membrane of the plasmatocytes and prevents plasmatocyte spreading by reducing cell volume through activating Na+ -K+ pump via protein kinase C signal pathway.