Excretion

Membrane dynamics of rapid fluid transportation in the malpighian tubules of the House cricket, Acheta domesticus

S. R. Hazelton, J. H. Spring, B. E. Felgenhauer & V. R. Townsend Jr

Dept. Biology, Univ. of Louisiana at Lafayette, Lafayette, LA 70504-2451, USA

In Acheta domesticus the Malpighian tubules (Mt) are composed of three morphologically distinct regions, each consisting of a single cell type. The bulk of the Mt is composed of the mid-tubule which is identifiable at both the light and electron microscopy level. The mid-tubule is 50-70 ?m in diameter and is composed of a single layer of cuboidal epithelial cells joined by septate desmosomes. These cells display the classic architecture of transport epithelia possessing extensive basolateral infoldings and a dense brush border. The mid-tubule is secretory in function and responds to endocrine stimulation. Our research centers on correlating the physiological response of the mid-tubule with the corresponding ultrastructural changes within the cells following the addition of various secretagogues. One of the most striking ultrastructural changes occurs when the Mt are stimulated with the second messenger dibutyryl cAMP. The increase in secretion rate (approximately doubling) is accompanied by notable changes in ultrastructure, the most prominent being membrane re-organization to increase the surface area of the basolateral infolds and vesiculation. The extensive pattern of endomembrane distribution noted in unstimulated tissue disappears following stimulation and the concretions (spherites) begin to dissolve. The area of the cell given over to vesicles/vacuoles more than doubles over 420 s post-stimulation. We followed the membrane dynamics of the mid-tubule using Transmission Electron Microscopy (TEM), Scanning Electron Microscopy (SEM) coupled with freeze-cleaving followed by extraction of the cytosol, and various fluorescent tracers (FM ® 4-64, ER-Tracker ™ , DiOC6 ). The SEM technique was developed in our laboratory to provide a three-dimensional view of membrane structure. Using this technique we were able to observe the increase in basolateral infoldings along with the presence of dilated areas of membrane suggestive of the vesiculation pattern of stimulated tissue previously observed with TEM. Our findings suggest that there is an intricate endomembrane network that vesiculates in response to rapid fluid transport possibly to compartmentalize fluid to prevent dilution of the cytoplasm. Supported by NSF grant IBN-9807948 to JHS and BEF.

Index terms: secretion, ultrastructure, cAMP

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