Fig. 2. (A) Elapsed time (seconds) necessary to release the hindgut (HG) content in in vitro preparations retaining different number of Malpighian tubules (MTs) undergoing an osmotic shock. Each bar represents mean ± s.e.m. of the elapsed time. Different letters (a–c) represent significant differences between samples in one of two experiments performed with similar results (N=3–6 samples per treatment; P0.05). (B) Aspect of the HG incubated with allatotropin (AT)-antiserum (1:100) undergoing an osmotic shock, after 3 h of the treatment (compare with the aspect of the HG under normal conditions in Fig. 1B). The same effect was obtained with three different preparations. (C) Elapsed time (seconds) until the beginning of the voiding in MTs–HG preparations retaining all MTs undergoing an osmotic shock, treated with different dilutions of the AT-antiserum [1:1000, 1:10 000, 1:100 000 and 1:1000 preadsorbed with 20 nmol of pure AT (P)]. Ctrl: samples without antiserum. Each bar represents mean ± s.e.m. of the elapsed time in one of two experiments performed with similar results (N=6 samples per treatment). Different letters (a–e) represent statistically significant differences between samples (P0.05). Note that preadsorbed samples are statistically different when compared with preparations treated with the same antiserum dilution (lower time), as well as with controls (higher). (D) Elapsed time (seconds) until the beginning of the voiding in MTs–HG preparations undergoing an osmotic shock. MTs were unattached and placed near the HG in the same diluted solution. Control, samples without antiserum; 1:1000, AT-antiserum dilution applied to the samples. Bars represent means ± s.e.m. ^*Significant differences between treatments (P0.05).