Membrane dynamics in the malpighian tubules of the house cricket, acheta domesticus.

In Acheta domesticus, the Malpighian tubules (Mt) are composed of three morphologically distinct regions (proximal, mid and distal), each consisting of a single cell type. The bulk of the Mt is composed of the midtubule, which shows the greatest response to corticotropin releasing factor-related diuretic peptides (CRF-DP). We know from previous laboratory studies that the second messenger cAMP and its analog dibutyryl cAMP (db-cAMP) cause an approximate doubling in the secretion rate and that this is accompanied by notable ultrastructural changes in the midtubule, especially membrane reorganization in the basal area and extensive vesiculation of the cytoplasm. In this study, we examined the morphological changes in membranes both at the cell surface and internally. By enzymatically removing the basal lamina, we examined the increase in spacing between infolded membranes initiated by db-cAMP stimulation. To examine the intracellular membranes, we used a technique developed for use in invertebrate tissues. This allowed the removal of the cytoplasm for high resolution scanning electron microscopy (HR-SEM) while maintaining the integrity of the lipid constituents of the cell. By using HR-SEM and confocal laser scanning microscopy (CLSM), we gained a unique three-dimensional perspective of the complexity of the internal membrane system of the A. domesticus Mt in both the unstimulated and db-cAMP-stimulated states.

Ultrastructural changes in the Malpighian tubules of the house cricket, Acheta domesticus, at the onset of diuresis: A time study.

The Malpighian tubules (Mt) of insects are responsible for maintaining osmotic homeostasis and eliminating waste from the hemolymph. When stimulated by diuretic factors the tubule cells are able to transport extraordinary volumes of fluid over short periods of time. We have been studying the changes that occur within the cells that accompany and facilitate this phenomenon. We present the ultrastructural changes that occur in the mid-tubule of the house cricket, Acheta domesticus, following exposure to the second messenger analog, dibutyryl cAMP, over the period from 15-420 sec. Vacuolation of the cytoplasm begins as early as 30 sec poststimulation with a significant increase in vacuolation occurring after 120 sec. As expected, there is an increase in the surface area of the basolateral membrane to facilitate the rapid movement of fluid into the cells. Other ultrastructural changes noted to accompany the onset of diuresis include the movement of mitochondria into areas adjacent to transport membranes, the vesiculation of Golgi, mobilization of CaPO(4) spherites, and a direct interaction of these spherites with active mitochondria. We discuss several possible roles for these changes in terms of rapid fluid transport.

Modes of control of insect Malpighian tubules: synergism, antagonism, cooperation and autonomous regulation.

Rates of fluid and ion secretion by insect Malpighian tubules are controlled by peptides, including CRF-related peptides and kinins, and in some species by serotonin. It now appears to be a general rule that tubule secretion rate is controlled through the interaction of two or more haemolymph-borne factors. In this review we suggest that these interactions may be classified as synergistic, cooperative, or antagonistic. When presented together, two diuretic factors may act in synergism, so that fluid secretion is stimulated to a greater extent than the sum of their individual effects. Synergism may involve one or more second messenger systems. Alternatively, diuretic factors may act in cooperation, so that although their overall effects are additive, cation and anion transport pathways are controlled separately by distinct second messenger systems. There is also one example of antagonism between factors controlling tubule secretion and between their respective second messengers; one factor is stimulatory, the other is inhibitory. In addition to the complex control of fluid and ion transport by haemolymph-borne factors, sophisticated autonomous regulatory mechanisms have been identified in Malpighian tubules. When triggered by appropriate stimuli, these mechanisms play homeostatic roles, preserving haemolymph osmolality or ionic composition.

Tests of potential adipokinetic hormone precursor related peptide (APRP) functions: lack of responses.

The adipokinetic hormone (AKH) precursor related peptides (APRPs) are end products of the synthesis of the well-conserved AKHs. The large amount of metabolic energy devoted to APRP synthesis suggests they have an important function(s) in the insects. Several functions have been proposed, but currently none are known. We tested whether the APRPs stimulate hyperlipemia, hypertrehalosemia, fat body glycogen phosphorylase activation, Malpighian tubule secretion, and hindgut myotropia. Surprisingly, none of these responses were stimulated by APRPs isolated from the lubber grasshopper, Romalea microptera (= guttata). In addition, the APRPs delivered in concert with AKHs did not significantly increase hyperlipemia, hypertrehalosemia, or phosphorylase activation over the AKHs alone. Our data discount several proposed functions for the APRPs. Arch.

Hormonal regulation in insects: facts, gaps, and future directions.

There are two main classes of hormones in insects: 1) the true hormones produced by epithelial glands and belonging to the ecdysteroids or juvenile hormones and 2) the neuropeptide hormones produced by neurosecretory cells. Members of these classes regulate physiological, developmental, and behavioral events in insects. Detailed accounts are given on isolation, identification, structure-activity relationships, mode of action, biological function, biosynthesis, inactivation, metabolism, and feedback for hormones involved in 1) metabolic regulation such as the adipokinetic/hypertrehalosemic peptides and the diuretic and antidiuretic peptides; 2) stimulation or inhibition of muscle activity such as the myotropic peptides; 3) control of reproduction, growth, and development such as allatotropins, allatostatins, juvenile hormones, ecdysteroids, folliculostimulins and folliculostatins, ecdysis-triggering and eclosion hormones, pheromone biosynthesis activating neuropeptides, and diapause hormones; and 4) regulation of tanning and of color change. Because of the improvements in techniques for isolation and structure elucidation, there has been rapid progress in our knowledge of the chemistry of certain neuropeptide families. With the employment of molecular biological techniques, the genes of some neuropeptides have been successfully characterized. There are, however, areas that are still quite underdeveloped. These are, for example, 1) receptor studies, which are still in their infancy; 2) the hormonal status of certain sequenced peptides is not clarified; and 3) functional studies are lacking even for established hormones. The authors plead for a concerted effort to continue research in this field, which will also advance our knowledge into the use of insect hormones as safer and species-specific molecules for insect pest management.

Differential effects of neuropeptides on the distal and mid-tubules of the house cricket.

In the Malpighian tubules of Acheta, the distal and middle segments are functionally and morphologically quite distinct (Spring and Kim, Mol Comp Physiol 12:130-145, 1993). Furthermore, they respond quite differently to corpora cardiaca (CC) homogenates, dibutyryl cAMP, and A23187 (Kim and Spring, J Insect Physiol 38:373-381, 1992). In this study we compared secretion by these two regions in response to Acheta and Romalea CC extracts, synthetic Manduca sexta diuretic peptide (Mas-DP1), and the family of synthetic myotropic peptides, the achetakinins, isolated from Acheta. Both Acheta and Romalea CC extracts had opposite effects on the two regions: mid-tubule secretion increased 3-fold whereas secretion by the distal segment declined 75-80%. Mas-DP1 increased secretion by the mid-tubule more than 3-fold and had no effect on the distal segment. All of the achetakinins decreased secretion by the distal tubule, with achetakinin 1 being least effective (55% inhibition) and achetakinin 5 being most effective (75% inhibition). Achetakinins 1 and 2 increased mid-tubule secretion by 3.7- and 3.3-fold, respectively, whereas the others had no effect on this region. Regarding HPLC fractions of CC extracts, in general the more hydrophilic fractions inhibited secretion by both distal and mid-tubules. The more hydrophobic fractions were nearly uniformly stimulatory when applied to the mid-tubule, and either inhibited secretion or had no effect on the distal region. The possible interpretations of these data and the implications towards future research are discussed.

Excretion in the house cricket: stimulation of rectal reabsorption by homogenates of the corpus cardiacum.

1. We describe an in vitro perfused preparation of Acheta domesticus rectum which allows direct comparison of Malpighian tubule secretion and rectal absorption under identical conditions. Rectal absorption is stimulated four- to sixfold by corpora cardiaca (CC) homogenates and the stimulated rate is sufficiently rapid to account for all the fluid secreted by the tubules. 2. The time course for increased fluid absorption is similar to that required to stimulate electrogenic chloride transport in locusts and grasshoppers. Chloride is rapidly absorbed by the rectum under all conditions, along with lesser amounts of Na+ and K+. Unlike the situation in locusts, K+ uptake is unaffected by CC homogenates and the stimulated absorbate is NaCl-rich, similar in composition to the NaCl-rich tubule fluid produced under stimulated conditions. The absorbate is always slightly hypo-osmotic to the perfusate, reaching a maximum differential of approximately 15 mosmol l-1 following CC stimulation. 3. The antidiuretic factor that reduces tubule secretion does not promote fluid reabsorption by the rectum.

A novel target for antidiuretic hormone in insects.

Diuresis in insects is controlled by two antagonistic hormone groups: diuretic hormones, which promote water loss, and antidiuretic hormones, which inhibit it. All known antidiuretic factors act solely to promote fluid reabsorption by the hindgut and do not affect secretion by the Malpighian tubules. In the house cricket, Acheta domesticus, an antidiuretic hormone was found that inhibits fluid secretion by the Malpighian tubules but has no effect on the hindgut. Correlations were found between the density of neurosecretory granules and the presence of antidiuretic hormone in the corpora cardiaca, suggesting that the hormone is released from specific axons. Its release is triggered by dehydration; the hormone is detectable in the hemolymph of water-deprived crickets. These results imply that an unusual mechanism regulates water balance in these insects.

Excretion in the house cricket (Acheta domesticus): ultrastructure of the ampulla and ureter.

An ultrastructural analysis of the ampulla and ureter of the cricket, Acheta domesticus, is presented. The excretory system of the cricket is unusual in that the 112 Malpighian tubules do not attach directly to the gut, but fuse to form a bladder-like ampulla which is joined to the colon by a muscular ureter. The ampulla consists of two cell types, primary and regenerative. Primary cells secrete large numbers of membrane-bound vesicles into the lumen and also appear to be involved in fluid reabsorption. Regenerative cells are very small and form a layer just beneath the basal lamina of the ampulla. They are believed to differentiate and replace sloughed off primary cells. The ureter is a muscular tube lined with cuticle which connects the ampulla (endoderm) with the colon (ectoderm). The probable origin and significance of the morphological modifications of the excretory system are discussed.

Excretion in the house cricket (Acheta domesticus): fine structure of the Malpighian tubules.

An ultrastructural analysis of the Malpighian tubules of the cricket, Acheta domesticus, is presented. The excretory system of the cricket is unusual in that the 112 Malpighian tubules do not attach directly to the gut, but fuse to form a bladder-like ampulla which is joined to the colon by a muscular ureter. The tubules have three structurally distinct segments and consist of four cell types. Attached by basal lamina to the outer surface of the distal tip are nodules, consisting of small cells on membranous stalks. These are presumed to serve as attachments to the body wall. The distal 20% of the tubule is hyaline, consisting of a monolayer of squamous cells that appear to be secretory. The mid-tubule comprises 75% of the total length and is the primary region for fluid secretion. It is also characterized by having large numbers of laminate spheres in the cytoplasm of the cells. The proximal 5% of each tubule consists of more columnar cells and may function in fluid resorption. The relationship between structural features and known physiological functions are discussed.