Diuretic factors controlling beetle Malpighian tubules : fluid secretion and immunohistochemistry

Abstract

Water balance in insects is under neuroendocrine control, and both diuretic and antidiuretic factors are thought to be involved. Despite being the largest and most diverse order of insects, the Coleoptera have been largely neglected in studies of hormonal control of excretion. However, the only insect from which both diuretic and antidiuretic peptides, acting on Malpighian tubules, have been isolated, is a beetle: the mealworm Tenebrio molitor. This study reports the effects of different diuretic factors on fluid secretion rates by mealworm tubules and briefly explores cross-reactivity of factors between beetle species, using the scarabaeid Onthophagus gazella. Attempts to localise an antidiuretic factor and a diuretic factor in the mealworm nervous system are also described. The study showed that calcitonin(CT)-like peptides from silkmoth and mosquito increased fluid secretion in mealworm tubules in a dose-dependent manner, but the cockroach CT-like peptide, Dippu-DH31, had no effect. The pharmacological agent thapsigargin, which mimics the action of kinins, caused a small but significant increase in tubule secretion rates. Since mealworm tubules are affected by more than one diuretic factor, the possibility of synergism between different factors was explored by testing CT-like peptides, thapsigargin and the mealworm corticotropin-releasing factor (CRF)-related diuretic peptide Tenmo-DH37, in various combinations and at different concentrations. No synergistic or additive effects were observed. C-terminal fragments of the locust and cockroach CRF-related diuretic peptides, Locmi-DH46 and Dippu-DH46 respectively, had no significant effect on fluid secretion. This is consistent with previous studies which have shown that the receptor binding and activation sites have to be linked in the same molecule for biological activity. However, the corresponding whole peptides increased fluid secretion, revealing the high degree of cross-reactivity of CRF-related peptides across insect orders. The mealworm diuretic factor Tenmo-DH37 increased fluid secretion in isolated tubules of O. gazelle in a dose-dependent manner, demonstrating cross-reactivity with this distantly related beetle species. However, brain homogenates from O. gazelle significantly inhibited fluid secretion in mealworm tubules. This is unusual, because extracts of insect nervous tissue have generally been shown to be a source of diuretic activity. Immunohistochemical techniques were used in an attempt to localise the mealworm antidiuretic factor Tenmo-ADFa in the nervous system of T. molitor. The antiserum developed against Tenmo-ADFa was found to be inadequately sensitive, and so these efforts were unsuccessful. The same techniques were employed in an attempt to locate the tissue containing a putative beetle CT-like peptide in the mealworm, using antiserum raised against the cockroach CT-like peptide Dippu-DH31. This too did not lead to a result. Experiments were repeated with various modifications, without success. It remains unclear whether the lack of results is due to degradation of the antiserum or if CT-like peptides are not present in detectable amounts in the mealworm nervous system.