Bioassay of Diuretic Hormone in Rhodnius

Abstract

The diuretic hormone of Rhodnius is a potent hormone released into the hemolymph of the insect within 1 min of the insect starting to feed (Maddrell and Gardiner 1976). Rhodnius is a bloodsucking insect, which at infrequent intervals takes very large meals of blood, each of which in the preadult stages can be up to 12 times as large as the insect itself. Not surprisingly, this raises many problems for the insect. Among them is how to allow for the great distension of the cuticular covering of the abdomen. This is achieved by the production of a plasticization factor (Maddrell 1966; see also Chapter 8 in this volume). A major problem is how to reduce the volume of the gut contents, which now contains an excessive volume of fluid. Since the quantity of salts and water in the gut is greater than needed, the insect can afford to excrete a large quantity of watery fluid, which is little more than a dilute solution of sodium chloride. This serves to concentrate the useful part of the gut content, reduce the size of the insect, and restore the osmotic concentration of the hemolymph (Maddrell and Phillips 1975). The speed of this process is impressive; it begins very promptly, as we have seen, and fluid is eliminated at a rate of up to 1.2 μl min^-1. Put another way, the insect can, in half an hour, eliminate a weight of fluid equivalent to its previous unfed weight. Such rapid excretion goes on for 2–4 h until 40-45% of the volume of the meal has been removed. An important element in this process is a great acceleration in the rate of fluid secretion by the insect’s Malpighian tubules. Each of the four tubules switches from secreting fluid at a rate of 0.1–0.5 nl min ^-1 to fluid secretion at up to 300 nl min^-1, around a thousand times faster. As has been known for some time (Maddrell 1962), the tubules are stimulated to do this by the appearance in the hemolymph of the diuretic hormone. The hormone is synthesized in two groups of neurosecretory cells found at the rear of the ganglionic mass in the mesothorax. It is now possible to isolate single cell bodies in a virtually uncontaminated state and estimate, by bioassay, their hormone content (Berlind and Maddrell 1979). It is the object of the next few pages to describe how the Malpighian tubules of Rhodnius are isolated and used to assay samples of tissue and hemolymph for substances capable of stimulating fast secretion by the tubules. The new methods for bioassay-ing single neurosecretory cell bodies are also described.