Histochemical studies of uric acid in some insects. 3. Excretion of uric acid by the malpighian tubules in Calliphora vicina and Rhodnius prolixus

In adult Calliphora uric acid is excreted throughout the Malpighian tubules. Histochemical preparations for the light microscope show uric acid passing through the cells and forming crystalline spheres in immediate contact with the microvilli. Uric acid appears to be synthesized and discharged into the haemolymph by the fat body cells. In Rhodnius there is no visible uric acid in the cells or lumen of the upper segment of the tubule (two-thirds of the total length of the tubule) apart from occasional deposits in the basal lamina. All uric acid excretion depends on the lower segment. Electron micrographs after argentaffin staining show high concentration of uric acid in the cytoplasm below the basal lamina (which also contains uric acid deposits). Uric acid is visible throughout the cell, particularly aroand the mitochondria; it is absent from the infolded plasma membrane and from all vacuoles. At the lumen there is a concentrated deposit of uric acid immediately beyond the plasma membrane. The uric acid particles unite with particles of unstained matrix material to form crystalline spheres. The fat body shows active synthesis of uric acid which is discharged by the cells into the intercellular channels and so to the basal lamina through which it passes into the haemolymph. As judged by histochemical preparations the haemolymph contains a high concentration of uric acid, very variable in different sites. Likewise large variations in uric acid secretion occur in different parts of the fat body.     

Subcellular localization of uric acid storage in the fat body of Manduca sexta during the larval-pupal transformation

The fat body of the tobacco hornworm, Manduca sexta, serves as the major site for uric acid storage during metamorphosis. Light and electron microscopic examinations of fat body stained with reduced silver to show the location of stored uric acid have revealed that most, if not all, fat body cells store uric acid. The extent of specific staining is proportional to the increase in uric acid concentration in fat body during the initial stages of metamorphosis. Storage is associated with discrete membrane-bound structures, designated as uric acid storage vacuoles. In larval fat body, the structures are round or elliptical-shaped vacuoles with electron-dense fibrous interiors and are about the size of observed mitocondria (0.5–1.0 μm). During the larval-pupal transformation, the storage vacuoles double in size and appear as fibrous cores with spaces between the cores and the surrounding membranes. Before pupal ecdysis, the storage vacuoles are concentrated around the nucleus of each cell but after that event they are more uniformly distributed within fat body cells.     

The transfer of lipid in insects from the epidermal cells to the cuticle

The structure of the pore canals and the tubular filaments they contain are described in a series of insects and types of cuticle. In all these cuticles the tubular filaments arise from the plasma membrane of the epidermal cells and they contain argentaffin material, regarded as sclerotin precursors, and lipid-staining material, regarded as wax precursors. These materials are transferred to the inner epicuticle and are exuded over the surface of the outer epicuticle to form the waterproofing layer as described in the preceding paper. They are also transported to those parts of the endocuticle destined to form hard exocuticle. There are no terminations of tubular filaments in the soft cuticle of Manduca larva, in the soft expanding cuticle of Rhodnius, and in the non-sclerotized post-ecdysial endocuticle of Tenebrio. Apis. etc. In the puparium of Calliphora lipid appears to be added by the epidermal cells directly and not by way of tubular filaments. It is confirmed that lipid is a component of sclerotized cuticle.     

Allantoin and allantoic acid titre in the faeces and tissues of the developing larva of the moth, Orthaga exvinacea Hampson

Allantoin and allantoic acid are investigated in the faeces and tissues of the developing sixth instar larva of the moth, Orthaga exvinacea. The nitrogen excreted as allantoin and allantoic acid is compared with nitrogen excreted as uric acid and ammonia. The larva excretes 2.35–5.14 μmol/g allantoin and 0.74–1.34 μmol/g allantoic acid which account for 0.83 to 2.39% and 0.23 to 0.53%, respectively, of the excreted total nitrogen. Allantoin and allantoic acid are found to be minor nitrogenous end-products of the larva. Allantoin and allantoic acid are also present in the haemolymph and fat body of the larva in varying concentrations. The level of allantoin in the haemolymph shows a negative correlation with the allantoin concentration of faeces and fat body. The allantoin is found to be stored in the fat body at a low level. The results of the present study also indicate the coexistence of uric acid storage and uricolysis.     

Demonstration of acid phosphatase activity induced by 20-hydroxyecdysone in the fat body of Calliphora

In the larval fat body of Calliphora erythrocephala, protein accumulation and autophagic activity occur prior to the onset of puparium formation. The involvement of the lysosomal system in the degradation of sequestered protein and cell organelles can be demonstrated by the electron-microscopical cytochemical localization of the lysosomal marker enzyme acid phosphatase in so-called protein granules. These granules contain not only newly synthetized or absorbed protein but also remnants of cell organelles such as mitochondria and endoplasmic reticulum. Ligation of the larvae behind the brain-ring gland complex prevents the appearance of these acid phosphatase-positive granules. They can be induced in ligated larvae by the injection of 20-hydroxyecdysone into the abdomen. These findings are briefly discussed in relation to the role of moulting hormones in normal development, especially with regard to the induction of autophagic activity.     

Histochemical studies of uric acid in some insects. I. Storage in the fat body of Periplaneta americana and the action of the symbiotic bacteria

An improved histochemical method for uric acid consists in precipitation as silver magnesium urate combined with fixation of the tissues in formol/glutaraldehyde followed by argentaffin reaction with silver nitrate buffered with "tris" to pH 9.5. This reveals urates both in solid deposits and in solution in the tissues. Polyphenols concerned in sclerotin formation also react. In Periplaneta, uric acid synthesized in the trophocytes is carried by intracellular and intercellular channels to form the intercellular deposits of solid spheres. The symbiotic bacteria in the mycetocytes in contact with the deposits appear to metabolize the uric acid and they disperse and eliminate the deposits.     

Fat body protein granules and storage proteins in the silkmoth, Hyalophora cecropia

Fat body cells of silkmoth pupae (Hyalophora cecropia ) contain granules, showing a less dense outer zone and a denser, often crystalline, inner portion appear after cocoon spinning and increase until the larval-pupal ecdysis; more granules are formed in females than in males. Urate granules, appearing fibrous in internal structure, first form about the same time, but their accumulation is more gradual, and continues in the pupa. Both types have been isolated by centrifugation. Protein granules dissolve in buffers to yield proteins 1 and 2, with distinct electrophoretic and antigenic properties. These proteins have been isolated individually from pupal fat body extracts by using their different thermal stabilities in phosphate buffer containing MgCl2 and (NH4)2SO4, respectively, and purification was completed by gel chromatography. Protein 1 has a molecular weight of 480,000 and a subunit of 85,000 daltons, while protein 2 gives values of 530,000 and 89,000, respectively. Their amino acid compositions are similar but distinct. Proteins 1 and 2 accumulate in the hemolymph, beginning 3 days before spinning, reach maximal levels at spinning, and then decline in the hemolymph while granules are formed in the fat body, although the total hemolymph protein concentration does not decline at this time. It is concluded that the fat body of the late, feeding larva synthesizes two related "storage proteins" and secretes them in partially crystalline granules as protein reserves for metamorphosis.     

Uric acid metabolism during pupal-adult development of Pieris brassicae

Uric acid metabolism has been investigated during the pupal and adult stages of Pieris brassicae. Uric acid and its main metabolite, allantoic acid, have been quantified in various organs (fat body, gut, wings) during development, in order to determine synthesis, degradation, and transport phenomena. Both labelling experiments (using 2-¹⁴C uric acid, guanine, and guanosine) and enzymatic studies (xanthine dehydrogenase, guanine deaminase, and uricase) were performed.Labelled uric acid, when injected into a young pupa, accumulates preferentially into the fat body, and its degradation leads to an increase in allantoic acid, which is found chiefly in imaginal structures (wings, heads, body wall). Since uricase is present only in low levels through the pupal stage, only a small fraction of uric acid is metabolized.In the developing pharate adult, uric acid is transported via the haemolymph from fat body to the wings and gut. Male wings accumulate more uric acid than female wings. At emergence, a large amount of uric acid and most of the allantoic acid are excreted into the meconium, but not together; uric acid is excreted into the so-called ‘meconium 1’ containing ommochromes, whereas its metabolite is eliminated only after wing expansion into ‘meconium 2’, a colourless fluid. Shortly before emergence, the fat body recovers its ability to synthesize uric acid, a fraction of which is excreted within ‘meconium 1’.During adult life, the synthesis of uric acid occurs in the fat body and ovaries, where it is especially abundant. Ageing organs (wings, heads, testes) accumulate it markedly. A small fraction is excreted together with allantoic acid by the butterfly.Purine catabolism pathways have been investigated, showing that in guanine derivatives, the freebase state of guanine leads quickly to uric acid (and its metabolites), whereas ¹⁴C-guanosine may be transformed into nucleotide and incorporated efficiently into wing pteridines when it is injected at the time of adult pigmentation.Another purine derivative, identified as adenosine, has been shown to accumulate in male fat body just before adult emergence. Its amount increases during the first days of emerged adult life, and it corresponds to an alternative pathway of purine catabolism. Its absence in females is related to development of the ovaries.     

Morphology and histochemistry of the adrenal medulla. I. Various types of primary catecholamine-storing cells in the mouse adrenal medulla.

Semithin sections (Araldite) of mouse adreno-medullary tissue were examined in the light microscope after perfusion fixation with glutaraldehyde, glutaraldehyde/formaldehyde or after freeze-drying followed by a treatment with hot formaldehyde gas. The following methods were employed: (i) aldehyde-induced fluorescence of catecholamines, (ii) Schmorl's ferric ferricyanide reaction, (iii) argentaffin reaction, and (iiii) staining with alkaline lead citrate followed by Timm's silver sulphide reaction. The correspondence of results obtained by the various methods was proven in consecutive sections or by successively applying different methods to identical sections. Four types of primary catecholamine-storing cells were identified. NA1 cells contain cytoplasmic granules up to 0.3 mum in diameter which stain black with ammoniacal silver and display a bright white to yellow fluorescence. NA2 cells show smaller cytoplasmic granules which stain brown with the argentaffin method and give white catecholamine fluorescence. NA3 cells appear yellow-earth after applying the argentaffin reaction and show greenish fluorescence. NA4 cells are hardly identified in the light microscope. These cells are significantly smaller than the above mentioned cells and characterized by a high nucleo-cytoplasmic ratio. They become straw coloured with ammoniacal silver and show greenish fluorescence. The argentaffin reaction was also used to identify these cells in semithin sections of glutaraldehyde/osmium tetroxide fixed material. The fine structure of the various noradrenalin-storing cells was studied in consecutive thin sections. NA1 cells were found to contain two populations of granules, the larger ones measuring between 300 and 350 nm, the smaller ones about 175 nm. The granules in NA2 cells correspond to this latter population (175 nm). NA3 cells contain an uniform granule population with a main diameter of 120 nm. The smallest granules are seen in NA4 cells being in the dimension of 80 nm. Granules in NA1 and NA2 cells show uniformly high density, whereas those in NA3 and NA4 cells display cores of varying density. Granules with moderately dense cores in NA3 and NA4 cells may represent partially emptied sites of noradrenalin storage or dopamin containing particles.     

The induction of lysosomal enzyme activity in the fat body of Calliphora erythrocephala: Changes in the internal environment

The activity of the lysosomal marker enzyme acid phosphatase in the larval fat body of Calliphora erythrocephala increases during development, but not at the same rate throughout the tissue. During the feeding stage, the posterior region has a higher acid phosphatase activity than the anterior region. When the larvae cease feeding on the 5th day of development, the acid phosphatase activity of the inactive anterior lobe increases rapidly in a mosaic-cell pattern. When 4-day-old feeding stage larvae are starved, this increase occurs one day earlier than normally. After the emptying of the gut, the acid phosphatase activity of all the anterior cells both in normal and in starved larvae exceeds that of those in the posterior region.Transplantation experiments indicate that the induction of acid phosphatase activity in the fat body during normal development, especially in the anterior region, is caused by a change in the internal environment when the larvae cease feeding. Both RNA and protein synthesis are involved in this induction process. Inductive factors are present in 5-day-old larvae as well as during formation of the puparium. The competence of the feeding-stage fat cells to develop high acid phosphatase activity is acquired before the actual induction takes place.     

MELANIN AND URATE ACT TO PREVENT ULTRAVIOLET DAMAGE IN THE INTEGUMENT OF THE SILKWORM,BOMBYX mori

The phenomenon that epidermal cells under the white stripes rather than black stripes contain many uric acid granules was found in larvae of several Lepidopteran species. However, the biological mechanism of this phenomenon is still unknown. In the present study, we take advantage of several silkworm (Bombyx mori) body color mutant strains to investigate the deposition patterns and biological mechanism of urate and melanin in the integuments of these mutant larvae. By imaging with transmission electron microscope, we found that there were some melanin granules in the larval cuticle in black body color mutant plain Black (p^B), but not in background strain plain (p) with white larval body color. In contrast, the larval epidermal cell of background strain had much more urate granules than that of black one. Furthermore, the uric acid content under the black stripes was significantly lower than that under the white stripes in a single individual of mottled stripe (p^S) with black and white stripes in each segment. Ultraviolet A (UVA) exposure experiments showed that the distinct oily (od) mutant individuals with translucent larval integument were more sensitive to the UVA damage than black body color mutant and background strain without any pigmentation in the larval cuticle. This is likely due to the absence of melanin granules and few urate granules in the integument of od mutant. Thus, both the deposited melanin granules in the cuticle and the abundant urate granules in the epidermis cells constitute effective barriers for the silkworm to resist UVA‐induced damage.     

意大利蜜蜂工蜂脂肪体胚后发育过程中细胞的增殖和凋亡

脂肪体是昆虫体内物质贮备和中间代谢的重要组织。本研究通过显微形态观察、 BrdU免疫组织化学和原位末端转移酶标记(TUNEL)细胞凋亡检测技术, 对意大利蜜蜂Apis mellifera ligustica工蜂脂肪体胚后发育过程中细胞的增殖和凋亡特点进行了比较研究。结果表明： 意大利蜜蜂工蜂脂肪体细胞数量的快速增加集中在幼虫发育前期（1-3龄）, 而细胞的凋亡则集中在蛹发育早期的2-3 d（预蛹-2日龄蛹）时间之内。在变态发育中, 工蜂幼虫脂肪体凋亡降解后重新组建形成成虫的脂肪体。本研究为昆虫脂肪体的功能研究以及昆虫组织细胞自噬和凋亡的机制研究提供一定的证据。     

INTRACELLULAR CHARACTERIZATION OF BEAN YELLOW MOSAIC VIRUS-INDUCED INCLUSIONS BY DIFFERENTIAL ENZYME DIGESTION

The granules which occur in the cells of a part of the midgut wall in Cercopid larvae and adults (Homoptera) have been studied by biochemical and cytochemical methods and by electron microscopy. The granules have a diameter up to about 2µ and contain calcium, magnesium, iron, carbonates, and phosphates. Protein and acid mucopolysaccharide have also been detected. A chromatographic study shows that uric acid and guanine are not present. The young concretions occur primarily in ergastoplasmic cisternae. They are first wholly electron-opaque, but their center becomes more and more clear. In very old spheres, only a thin shell of electron-opaque material remains. The spheres which have reached about 1µ in diameter are all associated with myelin figures. The granule-containing cells, which nearly occlude the lumen of the midgut in larvae, are eliminated in the very young adults, but the storage excretion still continue in adults.     

Mechanisms of black and white stripe pattern formation in the cuticles of insect larvae

Molecular mechanisms that produce pigment patterns in the insect cuticle were studied. Larvae of the armyworm Pseudaletia separata have stripe patterns that run longitudinally along the body axis. The pattern in the cuticle became clear by being emphasized by the increasing contrast between the black and white colors of the lines after the last larval molt. We demonstrated that dopa decarboxylase (DDC) mRNA as well as protein are expressed specifically in the epidermal cells under the black stripes. The pigmentation on the stripes was clearly diminished by injection of a DDC inhibitor (m-hydroxybenzylhydrazine) to penultimate instar larvae for 1 day before molting, suggesting that DDC contributes to the production of melanin. Further, electron microscopic observation showed that the epidermal cells under the gap cuticle region (white stripe) between the black stripes contain many uric acid granules, which gives a white color. Our findings suggest that the spatially regulated expression of DDC in the epidermal cells produces the black stripes while abundant granules of uric acid in the cells generate the white stripes in the cuticle. Based on these results, we concluded that this heterogeneity in the epidermal cells forms cuticular stripe patterns in the armyworm larvae.     

Argyrophile and argentaffin reactions in individual granules of enterochromaffin cells of the guinea pig

Summary Successive staining of cells of the enterochromaffin system of the guinea pig by an argentaffin and an argyrophile method shows that, as in man and rabbit, they can be divided into (a) those in which all granules are apparently argentaffin, (b) those in which all granules are argyrophile but none are argentaffin, and (c) those in which some granules are argentaffin (as well as argyrophile) while others are purely argyrophile. The presence of the third type makes it evident that no hard and fast line of distinction can be drawn between the so called argentaffin and argyrophile cells.I am grateful to Mr. Mohan Lal Sharma for help with the photographs and to Mr. Anand Parkash for technical assistance.     

Salivary gland function and chromosomal puffing patterns in Drosophila hydei

Summary The salivary glands of D. hydei larvae show differences between the cells in the distal (posterior) part and those of the proximal (anterior) part during the third instar. The first sign of these differences is an increase in cellular and nuclear volume in the distal cells of the gland, beginning at 103 hours after oviposition. After 125 hours the cytoplasm of the extreme distal cells acquires a reticulated structure, and at 130 hours these cells contain large granules or droplets of mucoprotein. From this moment up to puparium formation the number of cells containing these granules increases and the boundary of this type of cells shows a shift in the proximal direction. Just before puparium formation the granules disappear from the cells and a glue substance is secreted by the larvae. At this moment only a few cells in the extreme proximal part still lack granules. Electron-microscopical observations indicate that these cells were active in secretion, whereas all cells containing large granules are inactive in this respect during most of the third instar.During the early third instar a change in cell function occurs, i.e. from synthesis of substances presumed to be digestive enzymes which are secreted, to a synthesis of a glue substance which is stored. This change begins in the extreme distal cells of the gland.Investigation of the chromosomal puffing pattern revealed that a total number of 148 puffs were present during some period of the third instar, prepupal, and early pupal stages. The activity of 110 puffs was evaluated during a series of successive time intervals. Changes in the puffing pattern during puparium formation were compared with those observed during pupation.Proximal and distal nuclei differ in the activity level of a number of puffs, but only puff 47 B is restricted in activity to the distal cells. This puff becomes active at 119 hours and disappears 4 hours before puparium formation (156 hours). Determination of nuclear diameter and DNA in nuclei of both parts of the gland revealed a correlation between a particular DNA content and the function of the cell. Distal cells show higher nuclear diameters than proximal cells after the onset of granule production. The first differences in nuclear diameter can be seen at 103 hours. Cells in the transitional part of the gland, located between distal granulecontaining and proximal granule-negative cells, always show the same DNA content. These cells are found at different locations within the gland during the third instar. This zone of cells shows a shift in proximal direction during the third instar, identical to that of the neighbouring granule-containing cells.The possible interrelation between nuclear DNA content, the activity of puff 47 B, and the production of the glue substance were discussed.     

Argyrophile and argentaffin reactions in individual granules of enterochromaffin cells of reserpine treated guinea pigs

Summary The individual granules of enterochromaffin cells of normal and reserpine treated guinea pigs have been studied by staining slides of the duodenum first by an argentaffin method and subsequently by an argyrophile method. Some argentaffin cells can be shown to contain not only argentaffin granules, but also granules that are purely argyrophile. The relative number of such argentaffin cells is greatly increased following administration of reserpine, as depletion of their 5-hydroxytryptamine content converts argentaffin granules into purely argyrophile ones. On the basis of this finding it is confirmed that the argyrophile granule is merely an argentaffin granule depleted of its 5-HT content, and that the argyrophile (nonargentaffin) and the argentaffin cells represent different phases of a secretory cycle.This investigation was supported by a grant from the Indian Council of Medical Research. I am grateful to Ms. Ciba of India Ltd. for making available reserpine (Serpasil) and solvent for reserpine. It is a pleasure to thank Mr. Anand Parkash for technical assistance and Mr. M. L. Sharma for the photographs.     

Quantification of endocrine cells and ultrastructural study of insulin granules in the large intestine of opossum Didelphis aurita (Wied-Neuwied, 1826)

This study aimed to investigate the distribution of argyrophil, argentaffin, and insulin-immunoreactive endocrine cells in the large intestine of opossums (Didelphis aurita) and to describe the ultrastructure of the secretory granules of insulin-immunoreactive endocrine cells. Fragments of the large intestine of 10 male specimens of D. aurita were collected, processed, and subjected to staining, immunohistochemistry, and transmission electron microscopy. The argyrophil, the argentaffin, and the insulin-immunoreactive endocrine cells were sparsely distributed in the intestinal glands of the mucous layer, among other cell types of the epithelium in all regions studied. Proportionally, the argyrophil, the argentaffin, and the insulin-immunoreactive endocrine cells represented 62.75%, 36.26%, and 0.99% of the total determined endocrine cells of the large intestine, respectively. Quantitatively, there was no difference between the argyrophil and the argentaffin endocrine cells, whereas insulin-immunoreactive endocrine cells were less numerous. The insulin-immunoreactive endocrine cells were elongated or pyramidal, with rounded nuclei of irregularly contoured, and large amounts of secretory granules distributed throughout the cytoplasm. The granules have different sizes and electron densities and are classified as immature and mature, with the mature granules in predominant form in the overall granular population. In general, the granule is shown with an external electron-lucent halo and electron-dense core. The ultrastructure pattern in the granules of the insulin-immunoreactive endocrine cells was similar to that of the B cells of pancreatic islets in rats.     

Seasonal effects on cuticular permeability and epicuticular lipid composition inCentruroides sculpturatus ewing 1928 (Scorpiones: Buthidae)

Summary Third larval instar hemolymph of the fruitflyDrosophila hydei did not metabolize juvenile hormone (JH) at all developmental stages. In contrast, prepupal and pupal body fluid showed JH-esterase activity with a maximum at 4 h after puparium formation. In body wall and fat body of all developmental stages investigated, JH-metabolic activity was found. In both tissues JH catabolism was most active in the 120,000g supernatant and pellet. The 800g and 15,000g pellet showed a lower activity. In all subcellular fractions the JH-acid was identified as the predominant metabolite. There is evidence that JH-specific esterases are responsible for ester cleavage in the 120,000g supernatant. During mid and late third larval instar development in both body wall and fat body JH-esterase activity remains relatively constant.