Abstract
The female yellow fever mosquito Aedes aegypti deals with the volume and osmotic challenge from a blood meal by rapidly transporting excess fluid and ions across the epithelium and into the lumen of Malpighian tubules. Cyclic AMP is a second messenger in the pathway that mediates this process. Levels of endogenous cAMP increase in the MTs after a blood meal and, during in vitro studies, dibutyryl (db)-cAMP promotes fluid secretion by MTs. Transport across a plasma membrane is often associated with rearrangement of the actin cytoskeleton. Actin’s participation in the MT fluid secretion process is unknown, as are the effects of cAMP on the actin cytoskeleton. To determine whether a blood meal or db-cAMP cause rearrangement of actin, Malpighian tubules were stained either with an antibody that recognizes both F and G actin or with labeled phalloidin that stains just F actin. The tissue was viewed on a confocal microscope. Morphological and immunostaining intensity data of antibody stained tissue were recorded before and after stimulating secretion by a blood meal or db- cAMP. Both stimulants had similar effects. In untreated MTs, actin staining was present in the non-apical cytoplasm and, although less intense than in the apical region, was homogenous and visible. Following stimulation, a greatly decreased non-apical cytoplasm stain contrasted with an intense apical stain. This redistribution in the actin stain was both time and db-cAMP dose dependent. Db-cAMP had similar effects on actin distribution in larvae, pupae and adult MTs. Treating isolated MTs with chemicals that disrupt actin remodeling, namely cytochalasin D, phalloidin and latrunculin B, resulted in decreased fluid secretion and varied db-cAMP-stimulated actin stain redistribution. In tissue stained for just F actin, actin did not redistribute to the apical region of the cell. Instead, actin aggregates located in the nonapical region of the MT cells became larger after treatment with db-cAMP. Conclusion: Cyclic AMP has a marked effect on actin in the MT cells of both blood fed mosquitoes and isolated MTs. Events regulating fluid secretion in the Malpighian tubules such as increases in cAMP correspond with and result in both the redistribution of beta actin to the apical side of the cell and the formation of F actin bundles in the nonapical region of the MT cells. Actin redistribution is not dependent on actin’s ability to assemble or disassemble, nor is secretion wholly dependent on actin’s redistribution.