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.     

PROTEIN UPTAKE INTO MULTIVESICULAR BODIES AND STORAGE GRANULES IN THE FAT BODY OF AN INSECT

The fat body in Calpodes ethlius (Lepidoptera, Hesperiidae) takes up protein from the blood throughout the larval stage before pupation. Depending upon the phase of development, the protein appears in multivesicular bodies, in large storage granules, and in structures of intermediate form. There are three phases in the 8 days of the last larval stage; the first devoted to growth (molting to 66 hr), the second to synthesis for storage or export (M + 66 to M + 156 hr), and the third to preparation for pupation (M + 156 to pupation at M + 192 hr). From M + O to M + 156 and from M + 180 to M + 188 hr, protein is taken up into multivesicular bodies. Larger MVB's form a continuous series with the protein granules formed from M + 162 to M + 180 hr. Blood proteins increase in concentration and amount from M + 66 to M + 156 hr at the same time as the fat body cells have a high rate of incorporation of amino acids and a structure appropriate for protein synthesis. During granule formation, both amino acid incorporation and blood protein concentration decrease. Since foreign proteins injected into the blood appear in the granules, they are probably made mainly from sequestered blood. Protein uptake involves two stages: concentration between the cells, and ingestion in pinocytotic vesicles. The vesicles fuse to become MVB's or storage granules, depending upon their rates of growth and the addition of lytic enzymes. Since MVB's do not accumulate in the fat body and since many of them contain acid phosphatase and appear empty, they are presumed to be concerned in protein turnover.     

The effect of 20-hydroxyecdysone and protein on granule formation in the in vitro cultured fat body of Drosophila

The larval fat body of Drosophila melanogaster when cultured in a medium containing 20-hydroxyecdysone and foetal calf serum produces protein granules in the cytoplasm in a region-specific manner similar to that found in vivo. If ecdysteroid is omitted from this medium, the tissue continues to produce the granules at the same time, in the same region-specific manner, but in lower amounts. Only the high molecular weight fraction of the calf serum has the granule-inducing effect. Bovine serum albumin, herring protamine and bovine haemoglobin will also induce the granules to form. The degree of granule formation is directly proportional to the concentration of protein in the medium. Protein-free medium produces no granules, and protein concentrations in the medium above 3 mg/ml produce no further increase in granule formation. Although the medium containing foetal calf serum and 20-hydroxyecdysone induces more granule formation than medium containing only serum, the extent of granule formation does not differ at concentrations of hormone above 10^?6 M with any given concentration of serum. A minimal amount of serum (3.75%) permits measuring the effects of 20-hydroxyecdysone at concentrations below 10^?6 M. At this serum level the inducing effects of the hormone could be detected at concentrations of 10^?7 M.     

Pupal cuticle formation by Manduca sexta epidermis in vitro: Patterns of ecdysone sensitivity

During the larval-pupal transformation, various regions of the epidermis of Manduca sexta larvae have previously been found to require different lengths of exposure to the prothoracic glands in order to form pupal cuticle. To distinguish between requirements for differing threshold concentrations of ecdysone and those for differing durations of exposure to ecdysone, wandering stage larval epidermis was cultured in Grace's medium. When most of the thick larval cuticle was removed, the epidermis responded to concentrations of β-ecdysone of 1.0 μ/ml or greater for 4 days by forming cysts which later formed tanned pupal cuticle. No fat body or protein supplement was required. When the larval integument was explanted intact, similar requirements for cuticle formation and for tanning were found. All regions of the fifth abdominal segment required similar concentrations of β-ecdysone (0.4–0.6 μg/ml) for 4 days for 50% to form pupal cuticle, but gin trap epidermis required the least exposure to a threshold concentration of ecdysone (1.5 days in 0.9 μg/ml). The anterior dorsal intersegmental region required about 0.5 day longer, followed by the posterior intersegmental and the dorsal intrasegmental regions. Thus, the duration of exposure seemed more important. About 1 day longer of exposure to ecdysone was required for subsequent tanning of the new cuticle than for cuticle formation, yet tanning of the cuticle did not occur with prolonged exposure to ecdysone.     

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.     

Incorporation of 14C leucine into the fat body of the cockroach Periplaneta americana during oöcyte development

The fat body of Periplaneta americana incorporates labelled leucine into the protein during the period of oöcyte formation. The protein isolated from the ovary shows radioactivity only in the 20 hr treated animals; in the 1 hr treated animals the ¹⁴C activity is below background levels. The protein contents of the fat body and ovary were measured during the reproductive stages. The measurements indicate that the fat body may not store the protein which it synthesizes during reproduction.     

Induction of antibacterial activity in the haemolymph of the silkworm, Bombyx mori, by injection of formalin-treated Escherichia coli K-12 in the anterior and posterior body part of the ligated larvae.

1. Induction of antibacterial activity was investigated in the ligated fifth instar larvae of the silkworm, Bombyx mori, by injection of formalin-treated Escherichia coli K-12 into the haemocoel in the anterior and in the posterior body part, followed by activity determination by inhibition zone assay of the haemolymph at 12 and 24 hr after immunization. 2. At 12 hr after immunization, high antibacterial activity, approximately 6.8-7.5 mm in the anterior body part and 4.5-6.4 mm in the posterior body part in diameter of a clear zone (2.0 mm for no activity) was detectable in day 3 larvae. This result was in good contrast to expression of lectin gene in the ligated flesh fly, Sarcophaga peregrena (Shiraishi and Natori, 1988, FEBS Lett. 232, 163-166), in which only the anterior part of insect responded to stimulus of injury. 3. Antibacterial activity at 24 hr after immunization in days 3 or 4 ligated larvae was lower than that at 12 hr; 4.0-4.5 mm of activity was observed in the anterior body part, and no activity was observed in day 3 ligated larvae in the posterior body part. 4. Acid polyacrylamide gel electrophoresis of the haemolymph of immunized insects followed by overlay assay showed that the size of antibacterial activity bands were similar between the haemolymph from 12 hr and from 24 hr, and between the anterior and the posterior body part. This result was contradictory to the observation of activity by inhibition zone assay. The activity bands were associated with peptides that were similar to cecropin-like peptides A and B in the silkworm.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relationships between the parasitoid Hyposoter exiguae and Trichoplusia ni: Prevention of host pupation at the endocrine level

RNA synthesis in normal Trichoplusia ni fifth instars and hosts parasitized at ca. 12 hr post-ecdysis was followed by measuring ³H-uridine incorporation with an autoradiographic technique.Uptake of ³H-uridine was high in control prothoracic glands at 6 and 30 hr and their cytology indicated an active secretory phase which was most pronounced at 30 hr. At the same time, glands of parasitized larvae decreased incorporation and appeared less active than controls. At > 75 hr, control fat body cells incorporated almost no label but were filled with RNA-protein granules apparently sequestered from the haemolymph preparatory to pupation. With respect to incorporation and cytology, fat body of parasitized larvae was unchanged from earlier in the instar, which indicates that the changeover to pupal preparations had not taken place. Imaginal wing disks incorporated label and grew appreciably in control larvae but abruptly decreased uptake and showed no size increase in parasitized larvae. Incorporation of Malpighian tubule, midgut epithelium, and certain muscles at > 75 hr showed little change in parasitized larvae, but in controls activity was reduced and histolysis occasionally was evident in muscles.The parasitoid, Hyposoter exiguae, apparently prevented host larvae from pupating by preventing activation of host prothoracic glands in the fifth instar. Other tissues which are normally activated for metamorphosis by the prothoracic glands continued normal larval activities until the end of the association.     

Fine structure of the larval eyes of Mantispa sp. (Neuroptera : Planipennia,Mantispidae)

The stemmata of the first-instar larvae of Mantispa sp. (Neuroptera) were studied by scanning (SEM) and transmission (TEM) electron microscopy. These preparasitic larvae have a pair of anterior eyes and a single posterior eye on each side of the head. Each eye possesses an outer lens; beneath it, there is a well-developed crystalline body and a 3-tiered retina made up of a maximum of 12 sensory cells. The central fused rhabdom appears always to be composed of 4 sensory cells, each filled with pigment granules. The nuclear region shows Golgi bodies and abundant rough endoplasmic reticulum; the rhabdomeric regions contain vesicles, prominent multi-vesicular bodies and lysosomes. The eyes, whether double or single, are surrounded by a perineurium, to which muscle cells are attached.     

THE STRUCTURE AND FORMATION OF PROTEIN GRANULES IN THE FAT BODY OF AN INSECT

In the larva of the butterfly Calpodes ethlius, the fat body begins to store protein in the form of granules at about 30 to 35 hours before pupation, at a time when the endocuticle is being resorbed. At least two sorts of granule can be distinguished. The first granules to arise are those within vesicles of the Golgi complex. These may increase in size by incorporating material from microvesicles at their surface and by coalescence with one another. Later, at about 10 hours before pupation, another sort of granule arises by the isolation of regions of the endoplasmic reticulum (ER) within paired membranes derived from Golgi vesicles. Several of these ER isolation bodies coalesce, with fusion of their outer isolating membranes. The ribosomes and membranes may then disappear and the granules become indistinguishable from the protein granules formed from Golgi vesicles, or the ribosomes may remain and be embedded in dense crystalline protein, forming a storage body for both protein and RNA. Mitochondria are isolated within paired membranes in the same way as regions of the ER. The isolated mitochondria also coalesce in a similar manner. When the inner membranes are lost, the structure of a group of isolation bodies is indistinguishable from that of a cytolysome. Isolation within paired membranes, as described here, may be of general importance in segregating regions of massive lysis or massive sequestration.     

Ultrastructure and respiration of the fat body of diapausing and non-diapausing larvae of the corn borer,Diatraea grandiosella

A comparative study of the fat body of diapausing and non-diapausing larvae of the corn borer, Diatraea grandiosella, was undertaken using the electron microscope and the oxygen electrode. The electron microscopic results showed a shift from a synthetic to a storage function taking place in a 1 to 2 day period during the final instar of non-diapausing larvae, and in a 4 to 8 day period in that of pre-diapausing larvae. This transition was characterized by a decrease in the number of mitochondria and amount of rough endoplasmic reticulum, and by an increase in the number of proteinaceous granules and lysosomes. In vitro measurements using the oxygen electrode showed that the fat body is a normal aerobic respiratory tissue. The tissue reacted in a predictable manner to inhibitors of oxidative metabolism, including malonate, rotenone, oligomycin, and antimycin, and to the uncoupler, dinitrophenol. During the last instar the observed decrease in the respiratory rate of the fat body coincided with the observed ultrastructural changes in its cells. The fat body of 75 day old environmentally induced and juvenile hormone induced diapausing larvae consumed 90% and 78% less oxygen, respectively than that of 14 day old non-diapausing larvae.     

Storage function and ultrastructure of the adult fat body in workers of the ant Camponotus festinatus (Buckley) (Hymenoptera : Formicidae)

The presence of storage proteins in ants is perhaps most remarkable in its abundance in some species in the adult stage. The ultrastructure of fat body in workers of Camponotus festinatus (Hymenoptera : Formicidae) confirms that they do indeed store large quantities of protein, as well as lipid and carbohydrate, under some conditions. Rounded electron-dense granules, which are abundant in workers maintained in groups isolated from the parent colony, probably contain an arylphorin-like protein. Irregularly shaped electron-dense granules present in all workers regardless of age, caste or social environment, resemble primarily lysosomes, but lack acid phosphatase activity. Peroxisomes were also identified but were not associated with either type of dense granule. Lipid analysis showed that lipid storage followed similar patterns to protein storage, with isolated workers, especially soldiers, accumulating huge quantities of triglycerides. The relationship between storage of nutrient reserves and the presence of larvae suggests that the stores may function in regulating seasonal brood production.     

Ultrastructural observations on the organogenesis of the posterior silk gland of the silkworm, Bombyx mori

In the early stages of development (0 to 48 hr after organogenesis) the posterior silk gland cells of the silkworm, Bombyx mori, have characteristics of undifferentiated cells, that is, there are a number of free ribosomes in the cytoplasm and development of rough endoplasmic reticulum (rER) and Golgi bodies is very poor. Mitotic cells are frequently found. At ～ 60 hr when differentiation of the silk gland to the posterior, middle, and anterior divisions is completed, mitotic cells were no longer observable and the posterior silk gland is now composed of two rows of cells regularly packed forming a tubular structure. Differentiation of the cytoplasm is, however, not yet apparent and only a slight proliferation of rER can be observed. At 84 to 144 hr, proliferation of rER and transformation of rER from lamellar to vesico-tubular configuration are observed and Golgi vacuoles begin to enlarge. Just before hatching, the ultrastructures of cells are very similar to those of the later stage of the fifth instar when fibroin is synthesized extensively; the cytoplasm is filled with vesico-tubular rER, Golgi bodies, free secretory granules of fibroin, and mitochondria. Fibroin is probably synthesized, transported, and secreted in a manner similar to that in the fifth instar larvae.     

Fabrication of doughnut-shaped particles from spheroidal paramylon granules of Euglena gracilis using acetylation reaction

An anhydrous type of paramylon, the micro-sized granular storage carbohydrate (β-1,3-glucan) of Euglena, was transformed from a spheroidal to a doughnut-like shape by acetylation. Fourier transform infrared spectroscopic measurements suggested that the doughnut formation is due to removal of accessible regions of paramylon particles by acetylation of glucans. A time-course observation of the paramylon granules during acetylation by using field emission scanning electron microscopy revealed that the doughnut-making process begins with the removal of an outer membrane of the granule and that the central region of the granules is preferentially removed with the survival of a thick rim part to give the doughnut-like particles.     

Comparison of the localization of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Cry1A δ-endotoxins and their binding proteins in larval midgut of tobacco hornworm, <Emphasis Type="Italic">Manduca sexta</Emphasis>

Tobacco hornworm, Manduca sexta, is a model insect for studying the action of Bacillus thuringiensis (Bt) Cry toxins on lepidopterans. The proteins, which bind Bt toxins to midgut epithelial cells, are key factors involved in the insecticidal functions of the toxins. Three Cry1A-binding proteins, viz., aminopeptidase N (APN), the cadherin-like Bt-R₁, and membrane-type alkaline phosphatase (m-ALP), were localized, by immunohistochemistry, in sections from the anterior, middle, and posterior regions of the midgut from second instar M. sexta larvae. Both APN and m-ALP were distributed predominantly along microvilli in the posterior region and to a lesser extent on the apical tip of microvilli in the anterior and middle regions. Bt-R₁ was localized at the base of microvilli in the anterior region, over the entire microvilli in the middle region, and at both the apex and base of microvilli in the posterior region. The localization of rhodamine-labeled Cry1Aa, Cry1Ab, and Cry1Ac binding was determined on sections from the same midgut regions. Cry1Aa and Cry1Ab bound to the apical tip of microvilli almost equally in all midgut regions. Binding of Cry1Ac was much stronger in the posterior region than in the anterior and middle regions. Thus, binding sites for Bt proteins and Cry1A toxins are co-localized on the microvilli of M. sexta midgut epithelial cells.     

Ultrastructure and immunolocalization of digestive enzymes in the midgut of Podisus nigrispinus (Heteroptera: Pentatomidae)

The predatory stinkbug Podisus nigrispinus has been utilized in biological control programs. Its midgut is anatomically divided into anterior, middle and posterior regions, which play different roles in the digestive process. We describe the midgut ultrastructure and the secretion of digestive enzymes in the midgut of P. nigrispinus. Midguts were analyzed with transmission electron microscopy and the digestive enzymes amylase, cathepsin L, aminopeptidase and α-glucosidase were immunolocalized. The ultrastructural features of the digestive cells in the anterior, middle and posterior midgut regions suggest that they play a role in digestive enzyme synthesis, ion and nutrient absorption, storage and excretion. The digestive enzymes have different distribution along the midgut regions of the predator P. nigrispinus. Amylase, aminopeptidase and α-glucosidase occur in three midgut regions, whereas cathepsin L occurs in the middle and posterior midgut regions. The anterior midgut region of P. nigrispinus seems to play a role in water absorption, the middle midgut may be involved in nutrient absorption and the posterior midgut region is responsible for water transport to the midgut lumen.     

CYTOPLASMIC FINE STRUCTURE OF SCIARA SALIVARY GLANDS : I. Secretion

Cells from the anterior segment of the salivary glands of Sciara coprophila were found to synthesize and secrete into the gland lumen three morphologically distinct types of granule: 1) A large, electron-lucid granule, up to 1 µ in diameter, staining only faintly with pH 2 fast green and the PAS reaction; 2) an ellipsoid granule of moderate density, strongly fast green and PAS positive; and 3) a small spherical granule of high electron density. The cells contained numerous Golgi areas, up to an estimated 8,000 per cell. Evidence is presented for the transfer of material from the endoplasmic reticulum to the Golgi areas via small vesicles. Three types of Golgi areas were distinguishable, each containing intercisternal material resembling one of the three types of secretion granule. Patterns of secretion granule synthesis varied with the developmental stage of the larva as determined by counts of eye spots in the eye anlage. Lucid granules were most abundant in the youngest larvae, and decreased in abundance as larvae grew older, becoming virtually absent in prepupae. The small, dense granules were present in all gland cells, but became more prevalent in older larvae and prepupae. Ellipsoid granules were only occasionally present, and were independent of larval stage. It is suggested that lucid granules are digestive in function, since their abundance correlates with feeding patterns. Other granules may produce the external slime coating of the larvae, and also the mucoprotein component of the pupal cocoon.     

The induction of autophagy in isolated insect fat body by β-ecdysone

The fat body in Calpodes undergoes sequential organelle specific autophagy as a first step in the cell remodeling process necessary for metamorphosis to the pupa. This autophagy begins at about 36 hr before pupation and coincides with a critical period after which an isolated abdomen will pupate without further influence from the prothoracic glands. This suggested that autophagy might be induced by ecdysone. Fat body taken before the critical period and cultured in a medium containing β-ecdysone undergoes autophagy. Fat body from the same animal maintained in hormone-free medium retains the pre-critical period morphology with no autophagy. Autophagy is therefore directly induced by β-ecdysone. Fat body taken soon after the critical period continues with the autophagic sequence in hormone-free medium. Therefore the entire autophagic sequence is induced and does not require the continuing presence of hormone. Protein storage granule formation and cell dissociation, which occur in fat body at metamorphosis, are also induced by β-ecdysone.     

Potassium Loss during Galvanotaxis of Slime Mold

The posterior reticulated regions of the plasmodia of the slime mold, Physarum polycephalum, whose migration has been oriented by direct current (3.0 to 5.0 µa/mm² in the agar substrate), contain 30 per cent less potassium than the advancing non-reticulated region. The anterior regions have the same potassium concentration as that of the controls, approximately 32 meq/kg wet weight. Differences in potassium concentration between anterior and posterior regions of control plasmodia, not oriented by electric current, are less than 5 per cent. Sodium, in contrast to potassium, is generally less concentrated in the anterior than in the posterior regions of electrically oriented plasmodia, but sodium concentrations are extremely variable. No significant difference in protein concentration was found between oriented and control plasmodia. Thirty-five per cent of the total potassium, but none of the sodium, is found in acidified ethanol precipitates from plasmodial homogenates. Potassium, but not sodium, appears to be closely associated with processes which differentiate anterior from posterior in an oriented plasmodium.     

Microbial population dynamics during sludge granulation in an anaerobic-aerobic biological phosphorus removal system

The evolution of a microbial community was investigated during sludge granulation using a wide range of micro-scale and molecular biology techniques. Experimental results demonstrate that polyphosphate-accumulating granules were successfully cultured during the anaerobic/aerobic cycle. Improvement in sludge sedimentation performance occurred prior to the formation of granular sludge and was not affected by change in granule size. Rod-shaped and filamentous bacteria appeared to initiate granule formation and generate the structures that supported further granule growth. It was observed that mature granules supported microbial populations that differed from nascent granules and were predominantly packed with coccoid bacteria. It was further observed that the diversity of the granular microbial community increased as the granules grew. Accumulibacter, Nitrosospira and Thauera were mainly responsible for nutrient removal while microorganisms such as Rhodocyclus and Hyphomicrobiaceae appeared to be primarily responsible for forming and maintaining the granule structure.