The role of aquaporins in excretion in insects.

One of the aspects of insect osmoregulation that has most intrigued researchers is the ability of a simple tubular epithelium, such as the Malpighian tubule, to create both hypo- and hyperosmotic urine. Indeed, Ramsay's initial observation that isolated tubules could secrete a hypoosmotic urine led him to attribute the phenomenon to the active transport of water. In the ensuing decades several models for solute recycling have been proposed, but only in the last 15 years has it become clear that tubule water permeability is due to the presence of aquaporins (AQPs), the ubiquitous water transport proteins. There are 13 known human AQPs, and they are tissue and even membrane specific. It is now clear that the number and type of AQPs within a membrane are the major determinants of its water transport capacity. There are many gene homologs for the AQPs, so proof of function requires expression of the protein in a defined system. Within the insects, only seven AQPs have been functionally expressed and, of these, four directly or indirectly function in excretion. In this paper we review the basic structure and general function of AQPs and then examine the source, localization and functional attributes of those isolated from insects.

Localization of a Drosophila DRIP-like aquaporin in the Malpighian tubules of the house cricket, Acheta domesticus.

Malpighian tubules (Mt) are the primary excretory and osmoregulatory organs of insects, capable of rapidly transporting extraordinary volumes of fluid when stimulated by diuretic factors. In the house cricket, Acheta domesticus, the Mt are composed of three morphologically distinct regions (proximal, mid, and distal). Unlike the dipteran Mt, which have both primary and stellate cells, each region of the Acheta Mt consists of a morphologically uniform cell type. The mid and distal regions are both secretory in function and increase secretion rate in response to dibutyryl cAMP (cAMP). Achetakinin-2, while acting synergistically with cAMP on the mid-Mt, inhibits secretion by the distal Mt, and the effects can be reversed by cAMP. Using an antibody to the water-specific Drosophila aquaporin (DRIP), we demonstrated that DRIP-like immunoreactivity was found in both the distal and mid-Mt. The distribution of the aquaporin altered in response to stimulation and was consistent with the secretory data. The regulation of secretion in Acheta Mt is quite different from that of Drosophila, with both cation and anion/water transport occurring in the same cells. This is the first demonstration of the presence of an insect aquaporin, namely DRIP, in the Mt of an order other than the Diptera.

Morphology and ultrastructure of the Malpighian tubules of the Chilean common tarantula (Araneae: Theraphosidae).

Relatively little is known about the morphology and ultrastructure of the Malpighian tubules of spiders (Arachnida: Araneae). Our study represents the first investigation of the Malpighian tubules of a theraphosid spider and is the only study to examine the living Malpighian tubules using confocal laser scanning microscopy. In theraphosid spiders, the Malpighian tubules originate from the stercoral pocket in the posterior portion of the opisthosoma and extend forward toward the prosoma in a dendritic pattern. There are three distinct segments (initial, main, and terminal), all dark brown in appearance. Each segment has distinctive ultrastructural features. Both the terminal and the main segment appear to be composed of at least two cell types with finger-like cytoplasmic protrusions associated with one of these types. The terminal segment, which is most proximal to the stercoral pocket, is the largest in diameter. It is composed of large, cuboidal cells containing many mitochondria and lipid inclusions. The main segment is intermediate in diameter with many mitochondria and secretory vesicles present. The initial segment is relatively thin in comparison to the other segments and is intimately associated with the digestive gland. The cells of the initial segment contain very little cytoplasm, fewer mitochondria, secretory vesicles, and prominent inclusions.

Fluid-phase endocytosis does not contribute to rapid fluid secretion in the malpighian tubules of the house cricket, Acheta domesticus.

When the Malpighian tubules (Mt) of the house cricket (Acheta domesticus) are treated with dibutyryl adenosine 3', 5'-cyclic monophosphate (db-cAMP; 1 mM), which causes a doubling in secretion rate, more than 50% of the cell volume is occupied by vesicles within 420 sec of exposure. In view of the fact that the increase in vesiculation occurs concomitantly with stimulated fluid transport, we set out to determine whether the vesicles are formed as a result of fluid-phase endocytosis (pinocytosis) and subsequently used to transport fluid to the lumen as one means of increasing transport rate. We used fluorescent fluid-phase markers (Lucifer Yellow Carbohydrazide [LYCH] and Alexa 488 hydrazide) and an electron dense marker (cationized ferritin) to elucidate the degree of endocytosis that occurred with db-cAMP stimulation. We found that, although some fluid is taken into the cells of the mid-tubule via endocytosis, it does not coincide with the level of vacuolation present in stimulated tubules. The amount of LYCH transported into the primary urine by the db-cAMP-stimulated Mt decreased by 40% as compared to the unstimulated transport, and the rate of transport of LYCH was only 30% of the unstimulated tubules. In summary, our findings do not support the theory that the majority of the vesicles or vacuoles comprise intracellular, endocytotic compartments formed via a basolateral endocytotic pathway. We also found no evidence to support the functioning of vesicles or vacuoles as transcellular "shuttling" mechanisms to move fluid from the basal region to the apical membrane and into the lumen.

Excretion in the house cricket Acheta domesticus: Effects of diuretics on the structure of the mid-tubule.

Most structural studies of insect Malpighian tubules focus on freshly dissected tissue, which is in fact stimulated by diuretic factors. In this study, we examine tubules from the house cricket Acheta domesticus in four discrete secretory states: control (freshly dissected); unstimulated (held in vitro for 90 min prior to fixation); corpus cardiacum-stimulated (held in vitro for 60 min, then stimulated with corpus cardiacum homogenates for 30 min prior to fixation); and cAMP-stimulated (held in vitro for 60 min, then stimulated with dibutyryl cAMP for 30 min prior to fixation). In unstimulated tubules, we see a reduction in vacuolization and a near-complete collapse of the basolateral infolds. Stimulated tubules show several major structural shifts: mitochondria are darkly stained with well-defined cristae, there is extensive vacuolization of the tissue and expansion of the basolateral spaces, and the CaPO4 spherites appear to be ejected into the lumen. cAMP-stimulated tubules showed the most pronounced structural changes, including the presence of a newly reported ultrastructural feature for A. domesticus Malpighian tubules, referred to here as paracrystalline arrays, which appear as stacks of membrane localized in the perinuclear region. © 1996 Wiley-Liss, Inc.