Developmental studies on Malpighian tubule structure and function in Spodoptera Littoralis

Abstract

A study has been made on Malpighian tubule structure and function in Spodoptera littoralis. The concentrations of the main ions present in insect haemolymph were determined and a Ringer solution developed on the basis of this information. In vitro preparations of the medial region of the Malpighian tubules were set up to characterise fluid secretion using this normal' Ringer solution. Ion substitution experiments, involving varying concentrations of K+, Na+ and CI(^-) in the bathing media indicated that K+ and CI(^-) were necessary for normal fluid secretion rates to be maintained. Incubation in media containing high K+ (and zero Na+) yielded the maximum rate of fluid secretion in vitro, whereas high Na+ (and zero K+) resulted in a rate of fluid secretion lower than that of the control. CI(^-) substitution with gluconate resulted in reduced rates of fluid secretion .The agonists, 5-HT (10-3 m) and cAMP (10-3 M), both effected stimulation of fluid secretion; the maximal observed level of stimulation being 267.4% and 148.7%, respectively. In contrast, synthetic Manduca sexta diuretic peptide had no significant effect on the rate of fluid secretion by medial tubules of Spodoptera. The effects of known inhibitors of specific ion transport processes (ouabain; furosemide and NEM) on urine secretion was investigated. These three inhibitors inhibited fluid secretion significantly. NEM was the most effective inhibitor used; secretion being totally inhibited at 10(^-5) M NEM. Maximal inhibition with ouabain (1mM) and furosemide (10-(^3)m) was 94.6% and 80.3%, respectively. These results were taken as evidence for Na+/K+ ATPase , Na+/K+ /2C1(^-) co-transport and a V-type ATPase being involved in the transport of ions across tubule cells. Cytochemical localization studies showed the presence of Na+/K+ ATPase activity on the basal membrane of the proximal but not of the medial tubules. The apparent anomaly between this latter observation and die fact that fluid secretion by medial tubules was inhibited by ouabain is discussed. A hypothetical model has been constructed to explain ion and fluid secretion by the medial tubules of Spodoptera littoralis and its endocrine control. The rate of fluid secretion in vitro was studied at different ages throughout the 4th, 5th, 6th larval stages, the prepupae, the pupae and into adult life. It was shown that the rate of fluid secretion varied throughout development; both within each instar and between stadia. Fluid secretion ceased in the prepupal and pupal stages and started again following eclosion of the adult. Morphological studies on the relationship between the Malpighian tubules and the alimentary tract were carried out on last instar larvae, prepupae, pupae and adults; six tubules were present in all these stages. Each tubule consisted of several distinct regions; proximal, medial, iliac plexus, rectal leads and a cryptonephridial arrangement (larvae only). Electron microscopical studies on Malpighian tubules of 5th and 6th instar larvae and adults showed that one cell type was present in each region (the cryptonephridium was excluded from the E-M study). However, differences in cellular fine structure were apparent between regions. In the proximal region, the basal membrane foldings created a labyrinth and the apical membrane formed microvilli which were relatively short, loosely packed and contained mitochondria. In the medial and distal regions, which were similar in fine structure, the basal membrane was much infolded and extended further into the cytoplasm than in the proximal tubules. The apical microvilli of the medial region were considerably longer and more densely packed than in the proximal region and the apical surface was folded so that canaliculi were formed. Ultrastructural studies throughout development of the last larval instar revealed that the basic ultrastructure of the cells of the different regions of Malpighian tubules changed little. The major change seen during development from the last laraval stage to the adult was seen in the prepupal Malpighian tubules where the basal membrane foldings became very narrow and the apical microvilli were also very thin and their mitochondria were withdrawn into the cytoplasm. This change in the ultra structure was associated with cessation of fluid secretion. The tubule cells in the adult, unlike in the larva, contain numerous clear vacuoles and mineralized concretions. In addition the apical membrane of adult Malpighian tubule cells contained stalks which extend into the tubule lumen. The latter were only seen in larvae following treatment with die juvenile hormone analogue, methoprene.