Purification, characterization and mode of action of endogenous neuroendocrine factors in the forest ant, formica polyctena

Abstract

The Malpighian tubules of the forest ant, F. polyctena, have been studied for many years now by the research group physiology of the Limburgs Universitair Centrum (Diepenbeek, Belgium). In insects these tubules are responsible for the primary urine production. The latter is achieved via secretion, driven by the active transport of KCI from hemolymph to lumen, and passive diffusion of substances. Initially the transport mechanisms involved in this active transport have been investigated. In a subsequent phase the factors involved in the regulation of this active transport were investigated, i.e. the presence of endogenous factors with a direct effect on the primary urine production by the Malpighian tubules was investigated. Both diuretic and antidiuretic factors were found to be present in body extracts of the ants. Some of these factors, i.e. a diuretic peptide (Fop-DP I) and two myotropic peptides (Fop-TK I en Fop-TK II), could be purified. In the present study we tried to further complete this picture. Different factors, both diuretic (Fop-DP and Fop-GP) and antidiuretic (FopADF), were shown to be present in body extracts of the ant and some could be identified. The antidiuretic factor, purified from an abdomen extract, was shown to block the Transport mechanisms involved in the active transport of K+ through the cells of the Malpighian tubules (see chapter 1). The signal transduction pathway used by FopADF could not be clarified. The involvement of adenylate cyclase and / or an increase in intracellular cAMP could clearly be excluded (see chapter 2). A cross-reactivity of Tenebrio molitor Malpighian tubules towards FopADF could be demonstrated by Dr. S. Nicolson (see Note after chapter 1) and a similar antidiuretic factor was shown to be present in an abdomen extract of the honeybee, Apis mellifer (see chapter 4). The diuretic factors, both purified from head/ thorax extracts, were shown to be peptides. For Fop-DP approximately 35% of the amino acid sequence could be determined. Fop-DP acted via an increase in intracellular calcium (see Chapter 3). A Fop-DP related factor was also demonstrated to be present in a head extract of the honeybee (see chapter 4). The second diuretic factor, Fop-cGP, was fully characterized. It is a peptide of 30 amino acids which exerts its diuretic effect in Locust, via an increase in cAMP (see Chapter 6). The purification of Fop-cGP followed a preliminary study trying to establish the presence of CRF-related peptides in the forest ant. The latter were shown to be present in a head / thorax extract of the ant by means of chromatography and an ELISA based on polyclonal antisera to CRF-related peptides from other insects (see Chapter 5). Fop-cGP had the activity of a CRF-like factor but not the amino acid composition. Another factor having a CRF-like structure may be present but still awaits further purification and characterization. Below we tried to give a brief overview of the relevant results for each chapter. A table, added as bookmarker, summarizes the results and is intended as a reader guide. The factors involved in the endocrine regulation of the primary urine production of the forest ant is compared to the situation known for other insects.