Jean-Paul V. Paluzzi

Like other insects, secretion by mosquito Malpighian tubules (MTs) is driven by the V-type H + -ATPase (VA) localized in the apical membrane of principal cells. In Aedes aegypti , the antidiuretic neurohormone CAPA inhibits secretion by MTs stimulated by select diuretic hormones; however, the cellular effectors of this inhibitory signaling cascade remain unclear. Herein, we demonstrate that the VA inhibitor bafilomycin selectively inhibits serotonin (5HT)- and calcitonin-related diuretic hormone (DH 31 )-stimulated secretion. VA activity increases in DH 31 -treated MTs, whereas CAPA abolishes this increase through a NOS/cGMP/PKG signaling pathway. A critical feature of VA activation involves the reversible association of the cytosolic (V 1 ) and membrane (V o ) complexes. Indeed, higher V 1 protein abundance was found in membrane fractions of DH 31 -treated MTs, whereas CAPA significantly decreased V 1 abundance in membrane fractions while increasing it in cytosolic fractions. V 1 immunolocalization was observed strictly in the apical membrane of DH 31 -treated MTs, whereas immunoreactivity was dispersed following CAPA treatment. VA complexes colocalized apically in female MTs shortly after a blood meal consistent with the peak and postpeak phases of diuresis. Comparatively, V 1 immunoreactivity in MTs was more dispersed and did not colocalize with the V o complex in the apical membrane at 3 h post blood meal, representing a time point after the late phase of diuresis has concluded. Therefore, CAPA inhibition of MTs involves reducing VA activity and promotes complex dissociation hindering secretion. Collectively, these findings reveal a key target in hormone-mediated inhibition of MTs countering diuresis that provides a deeper understanding of this critical physiological process necessary for hydromineral balance.