Gut pH and amylase and protease activity in larvae of the New Zealand grass grub (Costelytra zealandica; Coleoptera: Scarabaeidae) as a basis for selecting inhibitors

Using a purpose-built antimony silver-silver chloride microelectrode we determined the pH of gut contents of field-collected third instars of the pasture pest Costelytra zealandica (White) (Coleoptera: Scarabaeidae) (grass grub). pH was 8.4 +/- 0.69 (mean +/- SD) (n = 7) in the anterior pre-caecal portion of the midgut; it rose to 10.8 +/- 1.28 (n = 19) in the anterior part of the remaining midgut, and 10.9 +/- 0.95 (n = 17) in the middle of the midgut, before dropping again to 9.3 +/- 0.68 (n = 19) at the posterior end of the midgut. The pH was 8.2 +/- 0.35 (n = 17) in the large hindgut. These data correct a previous suggestion that pH was approximately neutral in the grass grub gut. The data were then used to determine the functional localization of amylase and protease activities within the gut, by conducting assays at physiological pH values rather than the pH optima of the enzymes. Highest amylase activity was in the pre-caecal portion of the midgut; the hindgut showed relatively little activity. No activity was detected in the main part of the midgut because of that region's high pH. Protease activity was detected throughout the midgut. The pH data were also used to select putative inhibitors of the grass grub amylase, At pH 8.5 (the pH of the pre-caecal midgut), inhibitors from wheat and other Gramineae were active but a purified inhibitor from kidney bean showed no activity. These results indicate the importance of determining gut pH in studies of insect digestive enzymes.