Fig. 4. Whole Malpighian tubules were removed from animals and a modified Ramsay secretion assay employing two separate bathing droplets for the upper and lower segments of the tubules was used to collect secreted fluid. Ion-selective microelectrodes were used to measure the K⁺ and Cl^– concentration of the secreted fluid. Upper tubules were stimulated with 1 µmol l^–1 5HT and the lower tubules received 1 µmol l^–1 ZooneDH (A), 0.1 µmol l^–1 RhoprDH₃₁ (B), 0.1 µmol l^–1 leucokinin I (C,D), all three peptides at once (E) or 1 mmol l^–1 8-bromo-cyclic AMP (F; solid arrow in A–F). In addition, the lower tubules subsequently received 1 µmol l^–1 5HT (dashed arrow in A–F) as a positive control. A sample of the fluid from the upper tubule was taken at the end of the experiment by cutting the tubule at the junction of the upper and lower tubule (UMT). Values are means ± s.e.m. for A: N=4; B: N=5; C and D: N=6–11; E: N=7; F: N=6.