5-Hydroxytryptamine: a second diuretic hormone in Rhodnius prolixus.

Bioassays of 5-hydroxytryptamine (5-HT) in fifth-instar Rhodnius prolixus haemolymph using Calliphora salivary glands indicate that: (1) biologically active 5-HT is present, (2) in unfed animals there is not enough 5-HT to stimulate Malpighian tubule fluid secretion, and (3) there is enough 5-HT soon after the initiation of feeding to stimulate rapid tubule secretion. The 5-HT receptor antagonists ketanserin and spiperone reversibly and selectively inhibit 5-HT-induced fluid secretion, indicating the presence of specific 5-HT receptors on Rhodnius Malpighian tubules. The data provide evidence that 5-HT is a naturally occurring hormone acting with a previously described peptide hormone to regulate diuresis in this species.

Polymorphic organization of the endoplasmic reticulum of the Malpighian tubule. Evidence for a transcellular route.

Isosmotic fluid absorption carried out by many mammalian epithelia appears to be similar to the isosmotic secretion of insect epithelia such as the Malpighian tubules, which are responsible for urine formation and osmoregulation. We have studied by electron microscopy (80 kV) the three-dimensional characteristics of organelles in the Malpighian tubules of Rhodnius prolixus using thick sections (0.3-0.5 microns) and uranyl and lead impregnation. The ER presents a different organization in the upper (distal) and lower (proximal) segments of the Malpighian tubule. In distal secretory segment, the ER forms a network made of chains of vesicles having irregular shapes (ca. 0.06 micron in diameter) connected to each other by canaliculi while in the lower absorptive segment, the ER is made of parallel saccules arranged in stacks or whorls in the central region of the cytoplasm. In both segments, the ER network extends throughout the cytoplasm from the basolateral infoldings to the apex between the many mitochondria present in these two areas. A unique feature of these cells, revealed by thick sections, is the presence in each microvillus of either a mitochondrion or an ER canaliculus in continuity with the ER network. The ER does not seem to have any specific association with mitochondria or other organelles. As in the mammalian nephron, this ER organization is most likely related to specific segmental functions and adds support to its potential role as a transcellular epithelial route.

Sclerotin and lipid in the waterproofing of the insect cuticle.

In all the cuticles studied waterproofing is effected by extracuticular material, a mixture of sclerotin precursors and lipids, exuded from the tubular filaments of the pore canals. In Rhodnius larval abdomen it is a layer of thickness similar to the outer epicuticle, believed to be composed of 'sclerotin' and wax, in Schistocerca larval sternal cuticle and in Carausius sternal cuticle it is similar. In Tenebrio adult sternal cuticle of the abdomen, in both the extracuticular exudation and the contents of the distal endings of the tubular filaments, the wax component is obscured by hard 'sclerotin'. In Manduca larva a very thin layer of 'sclerotin' and wax is covered by an irregular wax layer, average 0.75 micron, twice the thickness of the inner epicuticle. In Periplaneta and Blattella the abdominal cuticle is covered by a soft waxy layer, often about 1 micron thick, which is mixed with argentaffin material. Below this is a very thin waterproof layer of wax and 'sclerotin' continuous with the contents of the tubular filaments, which is readily removed by adsorptive dusts. In Apis adult abdominal terga free wax plus sclerotin precursors form a thin layer which is known to be removed by adsorptive dusts. In Calliphora larva there is a very thin layer of the usual mixed wax and sclerotin and below this a thick (0.5 micron) layer, lipid staining and strongly osmiophil, likewise extracuticular and exuded from the epicuticular channels. This material (which is often called 'outer epicuticle') has the same staining and resistance properties as the true outer epicuticle on which it rests. In the abdomen of Calliphora adult the waterproofing wax-sclerotin mixture forms a thin layer over the entire cuticle including the surface of the microtrichia. There is also a thin detachable layer of free wax on the surface.

The distribution of lipid in the cell structure: an improved method for the electron microscope.

An improved partition method for visualizing lipid consists in fixing tissues in paraformaldehyde-glutaraldehyde followed by osmium tetroxide. Three progressive grades of lipid staining are then obtained: (i) by renewed osmium tetroxide alone, (ii) by partition in myrcene or farnesol solutions followed by renewed osmium, (iii) by saturated thymol in sucrose followed by partition and renewed osmium. No additional metallic stains are used. The thymol treatment in (iii) renders 'masked' lipid accessible to partition--the effect being regulated as required by time and temperature. Thymol used before the first osmium facilitates lipid extraction which provides a complementary test for lipid. The possibilities of the method have been demonstrated on sections of familiar tissues of insect (mainly Rhodnius, Hemiptera) and mammal (mouse). By and large the results support what is known already about the distribution of lipid in cells, but observations on lipid in muscle fibres, in the nucleolus and chromatin, in the cells of the adrenal cortex, in the lung and intestine suggest that the method might prove a source of new information.