A study of the concentrically laminated concretions, 'spherites' in the regenerative cells of the midgut of Lepidopterous larvae

Concentrically laminated granules, spherites, are sometimes found in the regenerative cells of midgut of some species of lepidopterous larvae. The spherites are formed in cytoplasmic vesicles just before ecdysis and disappear during the differentiation of the regenerative cells to columnar and goblet cells. They function as intracellular stores of compounds used in the growth of the cell. Phosphates of magnesium and perhaps calcium are probable constituents. Spherites are sometimes also found in the degenerating columnar cells where they are excreted into the lumen with the exfoliating epithelium. The phenomenon of periodic precipitation which is the physical-chemical basis of the formation of spherites is discussed.     

Polyphosphate polymers during early embryogenesis of Periplaneta americana

Inorganic polyphosphates (PolyP) are linear polymers of phosphate (Pi) residues linked by high-energy phosphoanhydride bonds. Despite a wide distribution, their role during insect embryogenesis has not been examined so far. In this study, we show the mobilization of PolyP polymers during the embryogenesis of the cockroach Periplaneta americana. PolyP was detected by enzymatic and fluorimetric assays and found to accumulate in two main sizes by agarose gel electrophoresis. Confocal microscopy showed their presence in small vesicles. In addition, X-ray microanalysis of small vesicles showed considerable amounts of calcium, sodium and magnesium, suggesting an association of PolyP with these elements. Variations of the free Ca⁺², Pi and PolyP levels were observed during the first days of embryogenesis. Our results are consistent with the hypothesis that phosphate ions modulate PolyP variation and that PolyP hydrolysis result in increasing free Ca⁺² levels. This is the first investigation of PolyP metabolism during embryogenesis of an insect and might shed light on the mechanisms involving Pi storage and homeostasis during this period. We suggest that PolyP, mainly stored in small vesicles, might be involved in the functional control of Ca⁺² and Pi homeostasis during early embryogenesis of P. Americana.     

Content,absorption quantities and intracellular storage sites of heavy metals in Diplopoda (Arthropoda)

By means of atomic absorption spectrophotometry, concentrations of more than 2500 mg kg^–1 Pb, 150 mg kg^–1 Zn, and 320 mg kg^–1 Cd could be detected in the intestine tissues of diplopods from a lead and silver smelter's spoil bank. While only small portions of the ingested lead and cadmium are absorbed in the midgut of these diplopods, the zinc uptake into the midgut epithelium reaches 33.8–37.5% of the zinc content in the food pulp when the animals were contaminated acutely. However, after long-term contamination with zinc, absorption and excretion of this metal balanced one another. Absorbed lead and cadmium are predominantly stored in the midgut cells of the diplopods; unspecific precipitation of heavy metal showed the spherites of the resorptive epithelial cells to be the main accumulation sites. Zinc is for the most part localized in or near the cuticle; electron energy loss spectroscopy and ESI electron spectroscopic imaging, however, showed this metal to be present also in the spherites of the midgut's resorption cells. These spherites are assigned to belong to the type A granule group since (i) they are concentrically structured, (ii) they are shown to contain great amounts of calcium and (iii) copper, a class B metal, could not be detected in these deposits.     

Effects of heavy metals on Tetraselmis suecica: Ultrastructural and energy-dispersive X-ray spectroscopic studies

The influence of metal contamination on the marine alga Tetraselmis suecica was investigated at physiological and ultrastructural levels. For this analysis, the growth response of this microalga was studied after the addition of various concentrations of heavy metals (Cd, Cu). The concentration corresponding to 50% growth inhibition (IC₅₀) and the number of days per cell cycle (Td) studied, revealed that the toxic effects of copper are heavier than those of cadmium. In the case of copper contamination, the Td grows with increasing metal concentration in the culture medium, while it remains unchanged during the cadmium contamination. The toxicity of cadmium, only observed in the latency phase of growth, suggests an adaptation phenomenon of T suecica to this metal. Ultrastructural changes in response to pollutants were investigated; copper induced cytoplasmic vacuolisation, organelle changes, appearance of cells with multilayered cell walls and excretion of organic matter. In the case of cadmium contamination, ultrastructural changes mainly affected the osmiophilic vesicles, of which both number and volume increased with increasing metal concentration in the culture medium. The results of X-ray microanalysis revealed that Cd and Cu were strongly present in excreted organic matter and osmiophilic vesicles. The latter can be excreted during cell division, thus participating in detoxification processes. Intracellular cadmium incorporation proved that some toxic effects of this metal are a result of interaction with endogenous cellular constituents. In the case of copper contamination, the presence of copper in walls of a multilayered cell suggests that these structures constitute an additionnal adsorbing area for this element, reducing metal free concentration in the medium. Mechanisms of metal detoxification of Tetraselmis suecica are discussed.     

Mineral congregations, “spherites” in the midgut gland ofCoelotes terrestris (Araneae): Structure,composition and function

Summary Spherites in the digestive and secretory cells of the midgut gland of the agelenid spiderCoelotes terrestris were studied by electron microscopy and histochemical methods. Spherites measured 1–6 m in diameter and were characterized by alternating layers of electron dense and electron lucent material. The main-components of spherites were calcium phosphates and calcium carbonates. Guanine and barium, as well as aminopeptidase and alkaline phosphatase were also present. The matrix consisted of proteins and carbohydrates. Numerous spherites were found together with excretory products within the excretory vacuoles of the digestive cells.Spiders fed with food containing lead, showed deposition of the metall in the spherites. It is then proposed that spherites, aside from their role in storing calcium and other ions, may function in detoxification of heavy metals.     

Cell differentiation in the embryonic midgut of the tobacco hornworm, Manduca sexta

While the larval midgut of Manduca sexta has been intensively studied as a model for ion transport, the developmental origins of this organ are poorly understood. In our study we have used light and electron microscopy to investigate the process of midgut epithelial cell differentiation in the embryo. Our studies were confined to the period between 56 and 95 hr of embryonic development (hatching is at 101 hr at 25 degrees C), since preliminary studies indicated that all morphologically visible differentiation of the midgut epithelium occurs during this time. At 56 hr the midgut epithelium is organized into a ragged pseudostratified epithelium. Over the next 10 hr, the embryo molts and the midgut epithelium takes on a distinctive character in which the future goblet and columnar cells can be identified. With further differentiation, closed vesicles in the goblet cells expand and subsequently communicate to the outside by way of a valve. The columnar cells form numerous microvilli on their apical surfaces that extend over the goblet cells. Both cell types form basal folds from a series of plasmalemmal invaginations. Differentiation occurs concurrent with a six-fold elongation of these cells.     

Yolk hydrolases in the eggs of Anticarsia gemmatalis hubner (Lepidoptera: Noctuidae): A role for inorganic polyphosphate towards yolk mobilization

Despite being the main insect pest on soybean crops in the Americas, very few studies have approached the general biology of the lepidopteran Anticarsia gemmatalis and there is a paucity of studies with embryo formation and yolk mobilization in this species. In the present work, we identified an acid phosphatase activity in the eggs of A. gemmatalis (agAP) that we further characterized by means of biochemistry and cell biology experiments. By testing several candidate substrates, this enzyme proved chiefly active with phosphotyrosine; in vitro assays suggested a link between agAP activity and dephosphorylation of egg yolk phosphotyrosine. We also detected strong activity with endogenous and exogenous short chain polyphosphates (PolyP), which are polymers of phosphate residues involved in a number of physiological processes. Both agAP activity and PolyP were shown to initially concentrate in small vesicles clearly distinct from typically larger yolk granules, suggesting subcellular compartmentalization. As PolyP has been implicated in inhibition of yolk proteases, we performed in vitro enzymatic assays with a cysteine protease to test whether it would be inhibited by PolyP. This cysteine protease is prominent in Anticarsia egg homogenates. Accordingly, short chain PolyP was a potent inhibitor of cysteine protease. We thereby suggest that PolyP hydrolysis by agAP is a triggering mechanism of yolk mobilization in A. gemmatalis.     

Cation-stimulated ATPase activity in purified plasma membranes from tobacco hornworm midgut

Purified goblet cell apical membranes from Manduca sexta larval midgut exhibit a specific ATPase activity approx. 20-fold higher than that in the 100 000 X g pellet of a midgut homogenate. The already substantial ATPase activity in this plasma membrane segment is doubled in the presence of 20-50 mM KCl. At ATP concentrations ranging from 0.1 to 3.0 mM, the presence of 20 mM KCl leads to a 10-fold increase in the enzyme's affinity for ATP. ATPase activity is greatest at a pH of approx. 8. In addition to ATP, GTP serves as a substrate, but CTP, ADP, AMP and p-nitrophenyl phosphate do not. Either Mg2+ or Mn2+ is required for activity and cannot be replaced by Ca2+ or Zn2+. The ATPase activity of goblet cell apical membranes is inhibited by neither the typical (Na+ + K+)-ATPase inhibitors, ouabain and orthovanadate, nor by the typical mitochondrial F1F0-ATPase inhibitors, azide and oligomycin. Although 1.5 microM DCCD is ineffective, 150 microM DCCD leads to total inhibition of ATPase activity. The ATPase activity of goblet cell apical membranes is stimulated not only by K+, but also, in order of decreasing effectiveness, by Rb+, Li+, Na+ and even Mg2+. Replacement of Cl- by Br-, F- and HCO3- has less influence than variation of the cations. However, replacement of Cl- by NO3- inhibits strongly this ATPase activity. The ATPase activity described above is characteristic of the alkali metal ion pump containing apical membranes of goblet cells and is not enhanced to a similar degree in other purified midgut epithelial cell plasma membrane segments. Its localization, its broad cation specificity and its insensitivity to ouabain all mimic properties of active ion transport by the lepidopteran midgut and suggest this ATPase as a possible key component of the lepidopteran electrogenic alkali metal ion pump.     

Detoxification of cadmium Ultrastructural study and electron-probe microanalysis of the midgut in a cadmium-resistant strain ofDrosophila melanogaster

Summary The midgut of a cadmium-resistant strain ofDrosophila melanogaster has been studied at the ultrastructural level and by electronprobe microanalysis (EPMA). Chronic exposure to cadmium leads to a concentration of the metal in a lysosomal system developed in both anterior and posterior segments of the midgut, where it coexists with copper and sulfur. This mechanism apparently ensures a permanent cadmium detoxification and prevents cellular injury. Wild-type flies fed on a cadmium-contaminated medium manifest the same detoxification process. As a result of contamination, copper is stored along the entire length of the midgut, including a part of the middle-midgut previously named copper-accuumulating region. Our data demonstrate that the midgut, particularly the posterior segment, is an accumulative organ for both cadmium and copper. The involvement of the metallothionein system in the detoxification process is discussed.     

The morphology and fine structure of the larval midgut of a moth (Manduca sexta) in relation to active ion transport.

Light and electron microscopic examination of the midgut of Manduca sexta has shown that the organization of this tissue is more complex than was originally believed. The midgut can be divided into anterior, middle and posterior regions on the basis of the pattern of folding of the epithelial sheet, and variations in the structure of goblet and columnar cells which occur along its length. The columnar cells show gradual structural changes form the anterior to the posterior end of the midgut. For example, the microvilli in the anterior region form a dense, interconnecting network from which vesicles break off. This organization becomes less obvious through the middle region, until by the posterior region each microvillus is unconnected to adjacent microvilli along its entire length and vesicles are no longer produced. Two distinct types of goblet cells are found. In the anterior and middle regions the goblet cells have a large basally located cavity, but in the posterior region the cavity occupies only the apical half of the cell. In both cases the cavity is formed by invagination of the apical membrane, which is studded with small particles implicated in active ion transport. In the anterior and middle regions this membrane is closely associated with mitochondria, but not in the posterior region. The significance of the observed structural differences is discussed in relation to active ion transport.     

Morphological regional differences of epithelial cells along the midgut in Diatraea saccharalis Fabricius (Lepidoptera: Crambidae) larvae

The sugarcane borer, Diatraea saccharalis Fabricius, is a pest to sugarcane and many other crops. This work aims to characterize morphological variability in the epithelial cells (columnar, goblet and regenerative) along the midgut of D. saccharalis larvae. Fragments of the midgut (anterior, middle and posterior regions) were fixed and processed by light and scanning electron microscopy. There are both cytochemical and ultrastructural differences in the morphology of the epithelial cells, depending on their localization along the midgut. The apical surface of columnar cells shows an increase in both number and size of the apical protrusions from the anterior to the posterior midgut regions. There is an increase in the amount of PAS-positive (Periodic Acid-Schiff Reaction) granules detected in the cytoplasm of both the columnar and regenerative cells, from the anterior to the posterior region. The goblet cell apical surface is narrow in the anterior region, and enlarged in the posterior midgut; the chamber's cytoplasm extrusion are small and thin at the apical cavity surface, being thicker, longer and more numerous at the basal portion of the cavity. Our results suggest that the sugarcane borer midgut has two morphologically different regions, the anterior and the posterior; the middle region is a transitional region.     

Calcification in insects: The dwelling-tube and midgut of machaerotid larvae (Homoptera)

The dwelling-tubes of machaerotid larvae consist of a mineralized organic scaffolding of mucofibrils. The mineral component accounts for 85 per cent of the dry weight and is composed of calcium, ferrous iron, manganese, magnesium, potassium, sodium, phosphate, carbonate, and chloride and of these the major ions are calcium and carbonate. Ferric iron in the form of ferritin is probably also present.Calcium, manganese, magnesium, and phosphate are derived from spherites secreted by a specialized region of the midgut. Calcium and phosphate are present in the spherites, probably as amorphous tricalcium phosphate. Subsequent to secretion the spherites are slowly dissolved and the calcium is incorporated into the dwelling-tube as calcium carbonate. Thus it appears that within the dwelling-tube calcium phosphate is converted to calcium carbonate.Ferritin and ferrous iron are secreted by another specialized region of the midgut and are also incorporated into the dwelling-tube.     

X-ray micronalysis of copper and sulphur-containing granules in the fat body cells of homopteran insects

Regions of the fat body of larvae of Chaetophyes compacta and Pectinariophyes sp. (Machaerotidae, Homoptera) which are closely associated with mycetomes have been analysed by electron probe X-ray microanalysis. It is shown that cells in these regions contain electron probe X-ray microanalysis. It is shown that cells in these regions contain electron dense granules which are rich in copper and sulphur. These two elements occur in the atomic ratio of 3:2 respectively. It is conjectured that copper may be bound to a sulphur containing metallothionein and that the granules represent either the end products of copper detoxification or serve as copper stores for synthesis of enzymes and macromolecules by the mycetomal symbionts.     

The ultrastructural investigation of the midgut in the quill mite Syringophilopsis fringilla (Acari,Trombidiformes: Syringophilidae)

The midgut of the females of Syringophilopsis fringilla (Fritsch) composed of anterior midgut and excretory organ (=posterior midgut) was investigated by means of light and transmission electron microscopy. The anterior midgut includes the ventriculus and two pairs of midgut caeca. These organs are lined by a similar epithelium except for the region adjacent to the coxal glands. Four cell subtypes were distinguished in the epithelium of the anterior midgut. All of them evidently represent physiological states of a single cell type. The digestive cells are most abundant. These cells are rich in rough endoplasmic reticulum and participate both in secretion and intracellular digestion. They form macropinocytotic vesicles in the apical region and a lot of secondary lysosomes in the central cytoplasm. After accumulating various residual bodies and spherites, the digestive cells transform into the excretory cells. The latter can be either extruded into the gut lumen or bud off their apical region and enter a new digestive cycle. The secretory cells were not found in all specimens examined. They are characterized by the presence of dense membrane-bounded granules, 2–4 μm in diameter, as well as by an extensive rough endoplasmic reticulum and Golgi bodies. The ventricular wall adjacent to the coxal glands demonstrates features of transporting epithelia. The cells are characterized by irregularly branched apical processes and a high concentration of mitochondria. The main function of the excretory organ (posterior midgut) is the elimination of nitrogenous waste. Formation of guanine-containing granules in the cytoplasm of the epithelial cells was shown to be associated with Golgi activity. The excretory granules are released into the gut lumen by means of eccrine or apocrine secretion. Evacuation of the fecal masses occurs periodically. Mitotic figures have been observed occasionally in the epithelial cells of the anterior midgut.     

Structure of the Malpighian tubule cells and annual changes in the structure and chemical composition of their spherites in the cave cricket Troglophilus neglectus Krauss, 1878 (Rhaphidophoridae,Saltatoria)

Periodical changes in the structure of spherites in the Malpighian tubule cells of the cave cricket Troglophilus neglectus were studied to elucidate their role during the cricket's life cycle in natural circumstances. Special interest was given to the dormant overwintering period when we hypothesized that the primary role of spherites is to supply minerals for basic vital processes. The investigation was carried out by light and transmission electron microscopy, energy dispersive X-ray spectroscopy, electron energy-loss spectroscopy and energy-filtering TEM. Spherites are present only in the middle Malpighian tubule segment, consisting of Type 1 cells, characterized, among other features, by a round, apically placed nucleus and numerous spherites, and a few Type 2 cells with an elongated nucleus in the centre and sparse spherites. At the beginning of dormancy in November juveniles, minerals are accumulated in spherites and then decline until March. In one-year-old May larvae, spherites are commonly rich in minerals, and from July onwards they are progressively exploited in the adults. Spherite destruction starts with apoptosis in senile October individuals. The findings suggest that the mineral supply of spherites in Malpighian tubules is crucial to supporting vital processes throughout the life cycle of T. neglectus.     

Calcium localization in the shell-forming tissue of the freshwater snail,Biomphalaria glabrata: a comparative study of various methods for localizing calcium

Summary The routes calcium might take across the mantle to the shell have been investigated with various electron-microscopical techniques in the freshwater snailBiomphalaria glabrata (Planorbidae, Basommatophora).In chemically-fixed tissue, calcium was precipitated with a tannic acid-antimonate technique in predominantly the intercellular spaces of the outer mantle epithelium and the interstitium below it. Some vacuoles of the outer mantle epithelium and one type of mucus cell in the inner mantle epithelium also contained precipitate. The presence of calcium in the precipitates was proved by electron energy loss spectroscopy combined with electron spectroscopic imaging. Incubation with lead acetate and uranyl acetate revealed binding-sites for calcium in the intercellular spaces of the epithelia interstitium and the mucus cells of the inner mantle epithelium. Precipitates were also seen after all incubations in the calcium spherites of the connective tissue.The concentrations of calcium and other elements were analysed in freeze-dried ultrathin sections of cryofixed mantle tissue by means of energy-dispersive X-ray microanalysis. Only in mitochondria of the musculature could high amounts of calcium and phosphorous be detected.     

INTRACELLULAR METAL COMPARTMENTALIZATION IN THE GREEN ALGAL MODEL SYSTEM MICRASTERIAS DENTICULATA (STREPTOPHYTA) MEASURED BY TRANSMISSION ELECTRON MICROSCOPY–COUPLED ELECTRON ENERGY LOSS SPECTROSCOPY1

Entry of metals in form of aerosols into areas of high air humidity such as peat bogs represents a serious danger for inhabiting organisms such as the unicellular desmid Micrasterias denticulata Bréb. ex Ralfs (Desmidiaceae, Zynematophyceae, Streptophyta). To understand cellular detoxification and tolerance mechanisms, detailed intracellular localization of metal pollutants is required. This study localizes the metals aluminum (Al), zinc (Zn), copper (Cu), and cadmium (Cd) in the green algal model system Micrasterias after experimental exposure to sulfate solutions by highly sensitive TEM‐coupled electron energy loss spectroscopy (EELS). Concentrations of the metals shown to induce inhibiting effects on cell development and cytomorphogenesis were chosen for these experiments. Long‐term exposure to these metal concentrations led to a pronounced impact on cell physiology expressed by a general decrease in apparent photosynthesis. After long‐term treatment, Zn, Al, and Cu were detected in the cell walls by EELS. Zn was additionally found in vacuoles and mucilage vesicles, and Cu in starch grains and also in mucilage vesicles. Elevated amounts of oxygen in areas where Zn, Al, and Cu were localized suggest sequestration of these metals as oxides. The study demonstrated that Micrasterias can cope differently with metal pollutants. In low doses and during a limited time period, the cells were able to compartmentalize Cu the best, followed by Zn and Al. Cu and Zn were taken up into intracellular compartments, whereas Al was only bound to the cell wall. Cd was not compartmentalized at all, which explains its strongest impact on growth, cell division rate, and photosynthesis in Micrasterias.     

Observations on the proventricular glands (‘organes racémiformes’) of the oribatid miteChamobates borealis (Acari,Oribatida): an organ of interest for studies on adaptation of animals to acid soils

The proventricular glands of the oribatid miteChamobates borealis (Trägårdh, 1902) were investigated by electron microscopy and histochemistry, and their function was tested in a laboratory experiment. Specimens of the same species collected during a field study also were investigated.The cells of the proventricular glands are characterized by great amounts of mineral spherites and seem to be sensitive to alterations of environmental pH and concentration of CaCO₃. It was demonstrated that a reduction of pH leads to a decrease of the spherites, whereas at a high pH the number of spherites increases.The proventricular glands with their spherites seem to play an important role in regulation of mineral budget, pH and detoxification of heavy metals.     

Histopathological effects of Bacillus thuringiensis on the midgut of tobacco hornworm larvae (Manduca sexta): Low doses compared with fasting

The cytology and ultrastructure of the midgut cells of Manduca sexta larvae are described for untreated controls, larvae which fed on a spore preparation of Bacillus thuringiensis, and larvae which were fasted for either 24 or 48 hr. New observations on the ultrastructure of midgut cells in Manduca larvae included the finding of specialized Golgi vesicles in anteriormost columnar cells and of regular arrays of expanded rough endoplasmic reticulum in goblet cells of the posterior midgut region. The present observations reveal that the columnar cells of the midgut responded cytologically in the same way to fasting as they did to exposure to the toxic spores of B. thuringiensis. The goblet cells, however, appeared unaffected by fasting but became swollen in response to feeding of B. thuringiensis spore preparation.