L-Glutamate retrieved with the moulting fluid is processed by a glutamine synthetase in the pupal midgut of Calpodes ethlius

Abstract

From apolysis until pupal ecdysis, the pharate pupa of the Brazilian Skipper ( Calpodes ethlius) lies wrapped in a prepupal shell composed of the larval cuticle and an ecdysial space (ES) filled with enzyme-rich moulting fluid (MF). In the 4 h before ecdysis the pharate pupa drinks the moulting fluid through its mouth and anus, and transfers the cuticular degradation products to its midgut (MG). At the same time, extra fluid passes across the body wall of the pharate pupa and flushes out the ES. The MF is recovered at an overall rate of 70 μl/h and reabsorbed across the pharate pupal midgut at about 26 μl/h. l-Glutamate was found to be the dominant amino acid in the moulting fluid. Total MF glutamate peaked at 850 nmol about 8 h before pupal ecdysis (P−8), but by ecdysis it had dropped to nearly zero as the MF became diluted with new fluid and was consumed. The drop in glutamate in the ES coincided with a rise in the glutamine content of the fluid in the midgut lumen. The highest rate of glutamine synthesis occurred in midguts isolated from pharate pupae actively drinking MF (P≤−4). The enzyme glutamine synthetase (GS) was found to be active in glutamate metabolism in the pharate pupal midgut. Glutamine synthesis in the midgut was l-glutamate-dependent and inhibited by two selective competitive inhibitors of GS activity, l-methionine sulfoximine (MSO) and glufosinate ammonium (GLA). Injection of GS inhibitors into the prepupal ES greatly reduced the glutamine content of the midgut epithelium by P+24. Although a corresponding increase in midgut glutamate levels was not seen, midgut serine levels in treated animals rose, suggesting that GS inhibitors shunted the MF-derived glutamate along an alternative metabolic pathway. GLA was much more toxic to pupae than MSO. Midgut GS appears to play a central role in the recycling of l-glutamate across the pupal MG epithelium at pupation.