Ultrastructure of osmoregulatory organs in larvae of the brackish-water mosquito,Culiseta inornata (Williston)

The ultrastructure of the Malpighian tubules, ileum, rectum, anal canal, and anal papillae of larvae of the mosquito Culiseta inornata was examined. The Malpighian tubules, rectum, and anal papillae have many of the ultrastructural features characteristic of ion transport tissues, i.e., elaboration of the basal and apical membranes and a close association of these membranes with mitochondria. The Malpighian tubules possess two cell types, primary and stellate. The larval rectum of C. inornata is composed of a single segment containing a homogenous population of cells. In this respect, the larval rectum of C. inornata is distinct from that of saline-water species of Aedes. The cells in the larval rectum of C. inornata, however, closely resemble those of one cell type, the anterior rectal cells, of the saline-water mosquito Aedes campestris with regard to cell and nuclear size, the percentage of the cell occupied by apical folds, and mitochondrial density and distribution. No similarities can be found between the rectum of C. inornata and the posterior segment of the saline-water Aedes, which functions as a salt gland. On this basis, we have postulated that the rectum of C. inornata does not function as a site of hyperosmotic fluid secretion. The ultrastructure of the anal papillae of C. inornata is consistent with a role in ion transport. The significance of these findings to comparative aspects of osmoregulatory strategies in mosquito larvae is discussed.     

The protocerebral neurosecretory system and associated cerebral neurohemal area of Acheta domesticus

Summary A comparison of rectal morphology and ultrastructure is made between a freshwater (A. aegypti) and salt water (A. campestris) species of mosquito larvae, and between A. campestris larvae producing hyper- and hyposmotic urine.The epithelium of A. aegypti contains one cell type characterized by infolding of both the apical and basal membranes, straight lateral borders, and evenly distributed mitochondria.The rectum of A. campestris contains distinct anterior and posterior regions, each made up of a single cell type. These two regions can be distinguished on the basis of cell thickness, depth of apical infolding and distribution of mitochondria. The anterior region is similar to the rectum of A. aegypti, while the posterior region is considered unique to the salt-water species and hence probably is associated with the formation of hyperosmotic urine.In A. campestris, the apical (rather than lateral or basal) membranes are probably the site of hyperosmotic urine production. Two possible mechanisms for this process are discussed.This work was supported by operating grants from the National Research Council of Canada.     

The anal portion as a salt-excreting organ in a seawater mosquito larva,A?des togoi Theobald

Summary Larvae of the marine mosquito,Aëdes togoi Theobald, tolerate environmental salinities ranging from fresh water to 300% sea water. When they were transferred from fresh water to sea water, sodium concentration in the haemolymph increased for the first 2 days and decreased to the seawater-adapted level within 4 days. When transferred from sea water to fresh water, the sodium concentration decreased markedly for the first 2 days and attained the freshwater adapted level after 4 days. When the larvae in sea water were ligated near the anus, they died within 3 days, showing an increased sodium level in the haemolymph. The larvae ligated at the neck lost considerable body weight and died within 4 days.When the anal portion, a terminal portion of the hindgut, was catheterized, the larvae maintained in sea water showed an increase in haemolymph sodium. The anal portion epithelium of the larvae adapted to 100 and 150% sea water demonstrated a strong positive reaction to the histochemical assay for chloride ions, whereas the reaction was negative or weakly positive in freshwater adapted larvae. In the larvae with the anal papillae ligated, a slight increase in haemolymph sodium occurred while in sea water. The anal papillae were weakly positive to chloride ions. Unlike salt-water mosquito larvae of the other species, in which the rectum is considered to be involved in hyperosmotic urine production and the anal papillae appear to be the extrarenal organ, the anal portion inA. togoi larvae seems to play an important role in excretion of excess ions when placed in hyperosmotic media.     

Osmotic regulation and salinity tolerance of the stonefly nymph, Paragnetina media

Nymphs of Paragnetina media were able to tolerate a range of salinities in the external media up 1.1% NaCl (～ 354 mOsm/l.) Nymphs maintained in distilled water showed excellent osmoregulation by producing hyposmotic urine. In a range of salinities, 0.4–0.95% NaCl, nymphs maintained their baemolymph concentration hyperosmotic to the external media. A breakdown in osmotic regulation occurred at 1.1% NaCl. There was no significant change (P > 0.01) in the oxygen consumption, indicating that metabolic energy demand for the osmoregulation was not affected.     

Freeze-fracture and tracer studies on the intercellular junctions of insect rectal tissues.

Both rectal pads of the cockroach and rectal papillae of the blowfly possess highly infolded lateral borders; these are associated by desmosomes and septate junctions that maintain the physical integrity of the cell layer at the luminal and basal intercellular regions. Adjacent cells are coupled by gap junctions that allow for cell-to-cell communication and which occur at intervals along the undulating lateral clefts. In rectal pads, occluding basal tight junctions are found as well as extensive scalariform junctions. The latter, like the stacked membrane infoldings of rectal papillae, exhibit intercellular columns and numerous intramembranous P face particles; these are undoubtedly involved in ion transport. In the inter-stack clefts of papillae, reticular septate junctions are encountered which, after freeze-fracture, possess a striking network of PF ridges and EF grooves that are discontinuous and not always complementary. These may serve to regulate the speed and extent of distension of the clefts during solute movement to allow for even and effective fluid flow in this transporting epithelium.     

An osmoregulatory syncytium and associated cells in a freshwater mosquito

In embryological terms the anal papillae are the product of eversion of the hindgut tissues. The rectum and the anal papillae have the same origin and have a marked structural similarity. The insect hindgut is very labile being able to produce salt transporting or ‘chloride cells’ from any of the tissues of which it is composed.The hindgut consists of four distinct regions: the ileum and part of the anal canal have a mechanical function, the rectum and the posterior anal canal contain transporting cells. Two new cell types, ‘interstitial’ and ‘tertiary’ are reported in the rectum.The structure of the anal papillae changes with increased salinity. Changes in the plasma membranes alter the surface area for transport. Changes in the number of mitochondria are not accompanied by changes in oxygen consumption. If mitochondria are the site of oxidative metabolism then their number docs not control the level of oxygen consumption.In Aedes aegypti the papillary epithelium appears to be a syncytium. Across the lumen of the papillae there are cellular sheets supporting the tracheoles. At the base of the papillae there is a cellular transition zone; circular muscles in this region may be used to occlude the papillae. The control of salt transport may be hormonal.     

Permeability of Alkali Metal Cations in Lobster Muscle : A comparison of electrophysiological and osmometric analyses

Single muscle fibers from lobster walking legs are effectively impermeable to Na, but are permeable to K. They shrink in hyperosmotic NaCl; they swell in low NaCl media which are hyposmotic or which are made isosmotic with the addition of KCl. In conformity, the membrane potential is relatively insensitive to changes in external Na, while it responds according to the Nernst relation for changes in external K. When the medium is made isosmotic or hyperosmotic with RbCl the volume and membrane potential changes are of essentially the same magnitudes as those in media enriched with KCl. The time courses for attaining equilibrium are slower, indicating that Rb is less permeant than K. Substitution of CsCl for NaCl (isosmotic condition) produces no change in volume of the muscle fiber. Addition of CsCl (hyperosmotic condition) causes a shrinkage which attains a steady state, as is the case in hyperosmotic NaCl. Osmotically, therefore, Cs appears to be no more permeant than is Na. However, the membrane depolarizes slowly in Cs-enriched media and eventually comes to behave as an ideal Cs electrode. Thus, the electrode properties of the lobster muscle fiber membrane may not depend upon the diffusional relations of the membrane and ions, and the osmotic permeability of the membrane for a given cation may not correspond with the electrophysiologically deduced permeability. Comparative data on the effects of NH₄ and Li are also included and indicate several other degrees of complexity in the cell membrane.     

The fine structure of epidermal papillae of Travisia forbesii (Annelida)

The structure of the epidermis of Travisia forbesii was described using light and electron microscopy. The epidermis is a highly modified variant of the normal one-layer polychaete epithelium. It consists of basal epidermal cells and an external layer of closely sited papillae consisting of glandular and supportive epidermal cells, and extensive electron-transparent intercellular spaces. The papillae are embedded in the thick cuticle. Each papilla has a peduncle, which is formed by one cell that penetrates the inner cuticle layer to the basal epidermal cells. A fold of basement membrane forms the core of the peduncle and ends in the base of a papilla. All epidermal cells are connected to each other with apical cell junctions and to the basement membrane with hemidesmosomes, so the epithelium is continuous and uninterrupted. The epidermis has an intra-epidermal neuron plexus. The structure of the papillae is compared with papillae and tubercles of other polychaetes, and the possible functional significance and phylogenetic implications of these structures are discussed.     

Temperature-Induced Inactivation of Cytoplasmic Biogel Osmosensing Properties is Associated with Suppression of Regulatory Volume Decrease in A549 Cells

Upstream intermediates of intracellular signaling involved in cell volume regulation remain poorly explored. Recently, we demonstrated that osmolarity-induced volume changes in permeabilized cells were several-fold higher than those observed with intact cells, indicating the osmosensing properties of cytoplasmic gel. To further examine the role of cytoplasmic biogel in cell volume regulation, we compared the action of short-term heat treatment on volume changes in intact and permeabilized A549 cells. Pretreatment of A549 cells at 48 °C suppressed swelling triggered by dissipation of Donnan’s equilibrium as well as by hyposmotic medium. Significantly, heat treatment completely abolished the action of hyposomotic medium on volume changes in permeabilized cells, showing that temperature elevation suppresses osmosensing properties via its effect on biogel rather than on plasma membrane water permeability. Identical heat treatment blocked the regulatory volume decrease (RVD) as well as the increment of Ba²⁺-sensitive K⁺-channel activity seen in control cells exposed to hyposmotic swelling. Unlike swelling, hyperosmotic shrinkage was decreased by twofold in cells subjected to 10-min preincubation at 50 °C. Our results disclose that osmosensing by cytoplasmic gel is a key event in the RVD triggered by hypotonic swelling. The role of biogel and plasma membrane in intracellular signaling triggered by hyperosmotic shrinkage should be further investigated.     

A MITOCHONDRIAL PUMP IN THE CELLS OF THE ANAL PAPILLAE OF MOSQUITO LARVAE

Mosquito larvae were raised to fourth instar in distilled water in order to maximally stimulate the salt-absorbing function of the anal papillae. Two exceptional features are observed, at the fine structure level, in the epithelial lining of the papillae. At the basal (cuticular) surface of the cells, the cell membrane is thrown into deep, narrow, parallel folds. The folds not only follow a rigid pattern at the cellular level but are also arranged at right angles to the long axis of the entire organ. A complicated pattern of canaliculi connects to the distal (plasma) surface of the cells. At this surface, paired mitochondria (sometimes triplets) are clamped about the membranes of the canaliculi to form structures referred to as "mitochondrial pumps." Mitochondria are also oriented in rather precise relation to the basal folds. Glycogen granules are found throughout the cytoplasm. The endoplasmic reticulum is sparse. The Golgi zones are flew and not well developed. Unidentifiable, irregular vesicles with lipid-like membranes are found associated with the basal folds. The distal (plasma) surfaces of the cells are covered by a homogeneous granular layer the composition of which is unknown.     

Larvicidal effect of pepper plants on Aedes aegypti (L.) (Diptera: Culicidae).

Ethanolic extracts derived from three species of the Piperaceae (pepper) family, Piper longum L., P. ribesoides Wall., and P. sarmentosum Roxb. ex Hunt., were evaluated for efficacy against early 4th instar larvae of Aedes aegypti mosquitoes using larvicidal bioassays. The highest larvicidal efficacy was established from P. longum, followed by P. sarmentosum and P. ribesoides, with LC50 values of 2.23, 4.06, and 8.13 ppm, respectively. Observations of morphological alterations on treated 4th instar larvae revealed that most organs, except anal papillae, had a normal structural appearance that was similar to controls. Under light microscopy, the internal structures of anal papillae in the treated larvae showed shrinkage, while the external features were normal in appearance. Ultrastructural studies, however, clearly demonstrated external destruction, with extensive damage and shrunken cuticle of the anal papillae. The structural deformation of anal papillae probably led to their dysfunction, which may be intrinsically associated with the death of the larvae. This study affords some evidence regarding the action site of the pepper extracts and suggests their potential in developing new types of larvicides used for mosquito control.     

Osmotic stress stimulates generation of superoxide anion by spermatozoa in horses

The objective of this study was to examine the interplay between osmotic and oxidative stress as well as to determine mechanisms by which osmotic stress increases superoxide generation in spermatozoa of horses. Superoxide production, as measured by dihydroethidium (DHE), increased when spermatozoa of horses were incubated under either hyperosmotic or hyposmotic conditions. This increase in superoxide production was inhibited by the MAP kinase p38 inhibitor, SB203580, and by the superoxide scavenger, tiron. Incubation of spermatozoa under hyperosmotic conditions increased overall protein tyrosine phosphorylation as measured by western blotting techniques; however, a similar increase was not detected when spermatozoa were incubated under hyposmotic conditions. The general protein kinase C (PKC) and protein tyrosine kinase (PTK) inhibitor staurosporine inhibited (P < 0.05) tyrosine phosphorylation in samples from cells under hyperosmotic conditions. In addition, the NADPH oxidase inhibitor diphenyleneiodonium (DPI) also inhibited (P < 0.05) protein tyrosine phosphorylation in cells under hyperosmotic conditions. In summary, these data indicate that incubation of equine spermatozoa under both hyposmotic and hyperosmotic conditions can increase superoxide anion generation. Under hyperosmotic conditions, this increased generation of superoxide anion was accompanied by increased protein tyrosine phosphorylation.     

Fine structure of the larval midgut of the fly Rhynchosciara and its physiological implications

The midgut of Rhynchosciara americana larvae consists of a cylindrical ventriculus from which protrudes two gastric caeca formed by polyhedral cells with microvilli covering their apical faces. The basal plasma membrane of these cells is infolded and displays associated mitochondria which are, nevertheless, more conspicuous in the apical cytoplasm. The anterior ventricular cells possess elaborate mitochondria-associated basal plasma membrane infoldings extending almost to the tips of the cells, and small microvilli disposed in the cell apexes. Distal posterior ventricular cells with long apical microvilli are grouped into major epithelial foldings forming multicellular crypts. In these cells the majority of the mitochondria are dispersed in the apical cytoplasm, minor amounts being associated with moderately-developed basal plasma membrane infoldings. The proximal posterior ventriculus represents a transition region between the anterior ventriculus and the distal posterior ventriculus. The resemblance between the gastric caeca and distal posterior ventricular cells is stressed by the finding that their microvilli preparations display similar alkaline phosphatase-specific activities. The results lend support to the proposal, based mainly on previous data on enzyme excretion rates, that the endo-ectoperitrophic circulation of digestive enzymes is a consequence of fluid fluxes caused by the transport of water into the first two thirds of midgut lumen, and its transference back to the haemolymph in the gastric caeca and in the distal posterior ventriculus.     

Fine structure of the pygidial glands of Bledius mandibularis (coleoptera : staphylinidae)

The pygidial glands of B. mandibularis produce a mixture of terpenes, fatty acid derivatives, and a benzoquinone. The morphology of these glands is described with particular attention to the ultrastructure of the secretory cells and their efferent ductules. Each functional secretory unit consists of two secretory cells (cortical and medullary) both of which are associated with a common extracellular cuticular ductule. The fenestrated tip of the ductule lies in a cavity bounded by the invaginated plasma membrane of the cortical cell; within the cavity surrounded by the medullary cell, the ductule is divided into a bulb region (where a spherical mass of fine cylinders surrounds the ductule itself) and an unfenestrated switchback region. Inflated cisternae of rough endoplasmic reticulum, filled with flocculent material of low electron density, are abundant in the cortical cytoplasm, and presumably represent primary secretory product en route to the cavity of this cell. The plasma membrane bounding this cavity is much infolded, and the inner surface of this membrane is studded with fine particles. In contrast, few cisternae are inflated in the medullary cell and the corresponding infolded plasma membrane is smooth. The manner in which both cells may cooperate to produce the heterogeneous secretory product is discussed.     

Research on the ultrastructure and function of the anal organ of the normal and lethal (l(3)tr) larvae of the Drosophila melanogaster

The anal organ of larvae of the wild type and the mutant ‘lethaltranslucida’ (l(3)tr, 3–20±0·8) of Drosophila melanogaster was studied by electron microscopy. By means of cryoscopy and microtitration the total osmotic concentration and the chloride content of the haemolymph were also determined. In addition, the effects of hypotonic and hypertonic solutions on the anal organs and the haemolymph concentration have been analysed in detail. We were especially interested in the functional significance and a possible disturbance of these anal organs in the lethal mutant.On the basis of the following characters the anal organ appears as a typical absorption organ: (a) The cuticle is distinctly thinner than that of the remaining body parts and has an enlarged surface by forming numerous porous infoldings. (b) On the cuticular surface the plasma membrane of the singlelayered epidermal cells forms a large number of folds oriented parallel to each other, resulting in a further increase of the absorption surface. (c) An extensive network of microtubules and a dense population of mitochondria, vacuoles, and vesicles in the cytoplasm suggest that an active transport process takes place in this organ.After 1 hr in a strong hypotonic solution (distilled water) the plasma membrane folds of both +/+ and l(3)tr larvae increase and penetrate deeper into the epidermal cell. The mitochondria also increase in number and are located between the apical folds. Conversely, in a hypertonic solution (1·42–11·96% NaCl) the plasma membrane folds become shorter and fewer in number. The mitochondrial density decreases. With increasing salinity and duration various unidentified bodies, degenerated mitochondria, and vesicles appear, indicating the beginning of autolysis.The osmotic concentration of 4-day-old +/+ larvae is found to be 1·02% NaCl, whereas that of l(3)tr larvae of a corresponding physiological age (5 days old) amounts to only 0·65% NaCl. Both genotypes are able to regulate the osmotic concentration in a hypotonic solution; the upper limit of salt concentration of the surrounding medium for a successful regulation is 5 per cent.The chloride concentration of +/+ larvae aged 4 days is found to be 0·19% NaCl, and that of l(3)tr larvae of a corresponding physiological age 0·14% NaCl. In a hypotonic solution both genotypes are capable of regulating the chloride concentration. However, this fails in a hypertonic medium with a concentration higher than 1% NaCl. When the anal organs are blocked by AgNO₃ impregnation, the regulatory ability breaks down completely. No distinct difference in the fine structure as well as in the regulatory achievement of the anal organs between the wild type and the l(3)tr mutant could be detected. It seems that the mutational effect does not lie primarily in a defect of the water balance.     

Hydromineral regulation in the saline water corixid Trichocorixa reticulata (Hemiptera: Corixidae)

The aquatic corixid Trichocorixa reticulata (Guerin-Meneville) inhabits coastal marshes, brackish water ponds and salt ponds of high salinity, suggesting the presence of well developed mechanisms for hydromineral regulation.Groups of corixids acclimated in salinities ranging from fresh water to just above 300% sea water (100‰) were analyzed for total body water content, haemolymph ionic and osmotic levels, and haemolymph free amino acids.Results indicate an excellent ability to maintain haemolymph Na⁺, Cl^?, Mg²⁺ and K⁺ hyperosmotic to the medium at low salinities and hyposmotic at high salinities. Calcium appears to conform closely to changes in external medium, becoming hyposmotic at very high salinities (80‰).Total haemolymph osmotic pressure was well regulated, the freezing point depression varying from 0.75°C in distilled water to 1.15°C in salinities of 100‰. Total body water was maintained at approx. 75% of the total animal wet weight at all salinities tested.Free amino acids were maintained between 40–60 mM in all tests and did not appear to change with salinity.     

Effect of extracellular osmolality on cell volume and resting metabolism in mammalian skeletal muscle

The purpose of the present investigation was to establish an in vitro mammalian skeletal muscle model to study acute alterations in resting skeletal muscle cell volume. Isolated, whole muscles [soleus and extensor digitorum longus (EDL)] were dissected from Long-Evans rats and incubated for 60 min in Sigma medium 199 (1 g of resting tension, bubbled with 95% O(2)-5% O(2), 30 +/- 2 degrees C, and pH 7.4). Medium osmolality was altered to simulate hyposmotic (190 +/- 10 mmol/kg) or hyperosmotic conditions (400 +/- 10 mmol/kg), whereas an isosmotic condition (290 +/- 10 mmol/kg) served as a control. After incubation, relative water content of the muscle decreased with hyperosmotic and increased with hyposmotic condition in both muscle types (P < 0.05). The cross-sectional area of soleus type I and type II fibers increased (P < 0.05) in hyposmotic, whereas hyperosmotic exposure led to no detectable changes. The EDL type II fiber area decreased in the hyperosmotic condition and increased after hyposmotic exposure, whereas no change was observed in EDL type I fibers. Furthermore, exposure to the hyperosmotic condition in both muscle types resulted in decreased muscle ATP and phosphocreatine (P < 0.05) contents and increased creatine and lactate contents (P < 0.05) compared with control and hyposmotic conditions. This isolated skeletal muscle model proved viable and demonstrated that altering extracellular osmolality could cause acute alterations in muscle water content and resting muscle metabolism.     

The function of anal papillae in salt adaptation of Drosophila melanogaster larvae

The anal papillae of Drosophila melanogaster larvae showed strong developmental plasticity in media with different osmotic pressure. The size of the papillae decreased with increasing salt content of the medium, which supports the hypothesis of its absorptive function. We showed that CI-transport at the site of the anal papillae decreased with increasing salt concentration, which also supports the hypothesis, that the function of anal papillae in osmoregulation is absorption at low salt content and not excretion at high salt content. The inactive area between the anal papillae increased with increased salt concentration, and thus contributed to the decreasing size of the anal papillae. Remnants of this area in pupae were mistakenly measured as anal papillae in a study on salt adaptation by Waddington (1959).     

Two non-exclusive strategies employed to protect Torulopsis glabrata against hyperosmotic stress

Several recent reports described an apoptosis-like programmed cell death (PCD) process in yeast in response to different environmental challenges. In this study, hyperosmotic stress caused by high NaCl concentration in culture medium induced cell death in the haploid yeast Torulopsis glabrata. Propidium iodide (PI) and PI/rhodamine-123 (Rh123) dual staining with flow cytometry showed that high salinity decreased intact cells by 16.5 %, increased necrotic cells by nearly twofold, and altered fermentative parameters appreciably. Morphological and biochemical indicators of apoptosis were apparent, specifically a decrease in mitochondrial membrane potential (?Ψ_m), translocation of phosphatidylserine (PS) from the inner to the outer side of the plasma membrane, generation of reactive oxygen species (ROS), and involvement of caspase all while plasma membrane integrity was maintained. Additionally, it was found that overexpression of YCA1 drastically stimulated cell death, indicating that activation of metacaspase might lead to cell death. However, T. glabrata growth under hyperosmotic stress was enhanced when FIS1, HOG1, and GPD2 were overexpressed, or when exogenous proline or glutathione (GSH) were added into the cultures, both of which could repress caspase-3 activity. Thus, in these concrete cases of overexpression of anti-apoptotic or anti-necrotic factors and pharmacological manipulations, it decreased T. glabrata cell death that might help to achieve higher fermentative efficiency.