Ultrastructure of Cotesia plutellae Bracovirus in Its Replication at Wasp Ovarian Calyx

Cotesia plutellae bracovirus (CpBV) is a polydnavirus symbiotic to an endoparasitoid, C. plutellae. Despite rich information on CpBV genome, there has been little known on its viral replication mode from proviral to episomal form. This study illustrates fine structures of the epithelial cells producing CpBV with a reference to non-producing ovarian epithelial cells. The ovarian epithelial cells of teneral females (within 12 h after emergence) were characterized by large nucleus and rich rough endoplasmic reticulum. CpBV particles were present only at the calyx region, in which follicle epithelial cells exhibited virogenic structures. Though a matured CpBV particle found in the calyx lumen was encapsidated in a single envelop containing multiple nucleocapsids, numerous free nucleocapsids were observed in the calyx epithelial cells and appeared to undergo assembly step to a final multiple capsid form. The multiple capsid forms appeared to be released into the oviduct lumen. The epithelial cells bordering the oviduct lumen showed phagocytosis presumably due to clearing cellular debris. At the calyx area close to the common oviduct, the epithelial cells appeared to maintain protein synthetic activity due to highly developed rough endoplasmic reticulum, but showed a marked decrease in the viral production.     

Particles containing DNA associated with the oocyte of an insect parasitoid

Viruslike DNA-containing particles are generated in the nucleus of cells located in the calyx region of the reproductive tract of the Hymenopterous parasitoid, Cardiochiles nigriceps. These particles are ca. 130 nm in diameter with a central core of DNA surrounded by an amorphous material enclosed by a unit membrane. These particles are secreted into the calyx lumen and compose the calyx fluid which is injected into hosts along with the eggs. The calyx fluid has been implicated in host regulation and in protection of the parasitoid from the immune response.     

Virus-like Particles in the Calyx of Phanerotoma flavitestacea (Hymenoptera: Braconidae) and Their Transfer Into Host Tissues

Virus-like particles occur in the calyx fluid and cells of the braconid parasitoid, Phanerotoma flavitestacea Fischer. These particles occur in the cytoplasm, nucleus, and cytoplasmic vacuoles of the calyx wall cells as well as in the lumen of the calyx. They were subsequently transferred to the host, Paramyelois transitella (Walker), during oviposition of the parasitoid. Within 2 hours after the wasps had oviposited, similar virus-like particles were found in the host in the cytoplasm of developing muscle tissue, fat body, and nerve tissue. Such particles were absent in unparasitized hosts. The significance of these findings as related to the avoidance of a host response is discussed.     

Microorganisms associated with the spider mite predator Metaseiulus (= Typhlodromus) occidentalis: Electron microscope observations

Two morphologically distinct forms of microorganisms were observed with the electron microscope in the tissues of the phytoseiid mite Metaseiulus (= Typhlodromus) occidentalis. The forms were distinguished on the basis of cell wall structure and cytoplasmic inclusions. One microorganism, designated type A, was observed in all mites examined. The second microorganism, type B, was observed in approximately two-thirds of the mites examined. If present, type B microorganisms occurred in all ovaries and egges, suggesting that transovarial transmission may take place. In some mites large numbers of type B microorganisms were observed within Malpighian tubule lumens and in a rectal plug. Whether this population was primarily or secondarily associated with a pathology is discussed.     

Nuclear secretory particles associated with the calyx cells of the ichneumonid parasitoid Campoletis sonorensis (Cameron)

Summary The present study is an ultrastructural investigation of the calyx region of the ichneumonid endoparasitoid Campoletis sonorensis. It appears that synthesis of electrondense secretory particles occurs within nuclei of calyx cells. The particles consist of an ovocylindrical electron-dense inner core and a surrounding unit membrane. After their formation the particles pass from the nucleus by budding through both membranes of the nuclear envelope. The particles, along with fully developed parasitoid eggs concentrate within the lateral oviduct lumen. Feulgen histochemical studies suggest the presence of DNA within the calyx fluid. The possible function of the particles is discussed.Approved for publication as TA 11744 by the Director, Texas Agricultural Experiment Station. Conducted in cooperation with the USDA and supported in part by Cotton, Inc. Grant CI—199 with funds made available through the USDA. Appreciation is also expressed to Sigma Xi for its contribution of funds during this study.     

The Impact of Microbial Biotransformation of Catechin in Enhancing the Allelopathic Effects of Rhododendron formosanum

Rhododendron formosanum is distributed widely in the central mountains in Taiwan and the major allelopathic compound in the leaves has been identified as (-)-catechin, which is also a major allelochemical of an invasive spotted knapweed in North America. Soil microorganisms play key roles in ecosystems and influence various important processes, including allelopathy. However, no microorganism has been identified as an allelochemical mediator. This study focused on the role of microorganisms in the allelopathic effects of R. formosanum. The microorganism population in the rhizosphere of R. formosanum was investigated and genetic analysis revealed that the predominant genera of microorganisms in the rhizosphere of R. formosanum were Pseudomonas, Herbaspirillum, and Burkholderia. The dominant genera Pseudomonas utilized (-)-catechin as the carbon source and catalyzed the conversion of (-)-catechin into protocatechuic acid in vitro. The concentrations of allelochemicals in the soil were quantified by liquid chromatography-electrospray ionization/tandem mass spectrometry. The concentration of (-)-catechin in the soil increased significantly during the extreme rainfall in the summer season and suppressed total bacterial populations. Protocatechuic acid accumulation was observed while total bacterial populations increased abundantly in both laboratory and field studies. Allelopathic interactions were tested by evaluating the effects of different allelochemicals on the seed germination, radicle growth, and photosynthesis system II of lettuce. Protocatechuic acid exhibited higher phytotoxicity than (-)-catechin did and the effect of (-)-catechin on the inhibition of seed germination was enhanced by combining it with protocatechuic acid at a low concentration. This study revealed the significance of the allelopathic interactions between R. formosanum and microorganisms in the rhizosphere. These findings demonstrate that knowledge regarding the precise biotransformation process of (-)-catechin by microorganisms in the environment is necessary to increase our understanding of allelopathy.     

Improvement of bacterial transformation efficiency using plasmid artificial modification

We have developed a method to improve the transformation efficiency in genome-sequenced bacteria, using ‘Plasmid Artificial Modification’ (PAM), using the host''s own restriction system. In this method, a shuttle vector was pre-methylated in Escherichia coli cells, which carry all the putative genes encoding the DNA modification enzymes of the target microorganism, before electroporation was performed. In the case of Bifidobacterium adolescentis ATCC15703 and pKKT427 (3.9 kb E. coli-Bifidobacterium shuttle vector), introducing two Type II DNA methyltransferase genes lead to an enhancement in the transformation efficiency by five orders of magnitude. This concept was also applicable to a Type I restriction system. In the case of Lactococcus lactis IO-1, by using PAM with a putative Type I methyltransferase system, hsdMS1, the transformation efficiency was improved by a factor of seven over that without PAM.     

Survival kinetics of lactic acid starter cultures during and after freeze drying

Survival kinetics of lactic acid starter cultures were modeled considering the microorganism and external medium interfacial area as the critical factors determining the resistance of the microorganisms to freeze-drying. Surviving fraction of the microorganisms increased with the increasing biomass concentration during freeze-drying, and this is attributed to the mutual shielding effect of the microorganisms against the severe conditions of the external medium. Survival of the microorganisms over the storage period after freeze drying was enhanced by the presence of dead microorganisms which reduce the interfacial area between the live cells and the external medium. Streptococcus thermophilus was found to be more resistant to freeze-drying conditions than Lactobacillus bulgaricus. Storage under vacuum or nitrogen was superior to storage under air. Poor survival rates under air was attributed to oxygen diffusion into the dry cells through the interfacial area.     

Allosteric regulation of tRNA import: interactions between tRNA domains at the inner membrane of Leishmania mitochondria

Import of nucleus-encoded tRNAs into the mitochondria of the kinetoplastid protozoon Leishmania involves recognition of specific import signals by the membrane-bound import machinery. Multiple signals on different tRNA domains may be present, and further, importable RNAs interact positively (Type I) or negatively (Type II) with one another at the inner membrane in vitro. By co-transfection assays, it is shown here that tRNA^Tyr (Type I) transiently stimulates the rate of entry of tRNA^Ile (Type II) into Leishmania mitochondria in transfected cells, and conversely, is inhibited by tRNA^Ile. Truncation and mutagenesis experiments led to the co-localization of the effector and import activities of tRNA^Tyr to the D domain, and those of tRNA^Ile to the variable region–T domain (V-T region), indicating that both activities originate from a single RNA–receptor interaction. A third tRNA, human tRNA^Lys, is imported into Leishmania mitochondria in vitro as well as in vivo. This tRNA has Type I and Type II motifs in the D domain and the V-T region, respectively, and shows both Type I and Type II effector activities. Such dual-type tRNAs may interact simultaneously with the Type I and Type II binding sites of the inner membrane import machinery.     

The effect of growth state on the activity of the protein kinase isoenzymes an increase in type II cyclic AMP-dependent protein kinase is correlated with growth arrest in G1 phase

Native polyacrylamide gels have been used to resolve protein kinase isoenzymes from cultured cells and the protein kinases have been identified by carrying out phosphorylation reactions in the gel. Following electrophoresis, the gels were incubated with histome and [γ-³²P]ATP. The gels were then thoroughly washed and dried down, and the protein kinases were located by autoradiography. Protein kinase activity as measured in the gel system was a linear function of cytosol protein concentration up to about 100 μg per channel and incorporation of ³²P into histone was time dependent. Three bands of protein kinase activity were resolved in cytosol samples from baby hamster kidney (BHK) fibroblasts. The band with the lowest relative mobility utilized histone IIA or casein equally well as substrate protein whereas bands 2 and 3 demonstrated a clear preference for histone. Bands 2 and 3 displayed a relative mobility in electrophoresis that was identical to that observed for cyclic AMP-dependent protein kinases I and II from rat liver. Treatment of cyctosol samples with cyclic AMP prior to electrophoresis resulted in the disappearance of cyclic AMP-dependent protein kinases from the gel profile. This method was employed to identify bands 2 and 3 as cyclic AMP-dependent protein kinases. The protein kinases in growth-arrested cells were compared with proliferating cells. We have observed a 3.5-fold increase in the activity of Type II protein kinase as the cells arrest growth in G₁ phase of the cell cycle. This increase in Type II is correlated with the increase in cells blocked in G₁ and a decrease in II Type activity appears to be an early event in permitting cells to leave G₁ and resume growth.     

Ecdysteroid-titre reduction and developmental arrest of last-instar Heliothis virescens larvae by calyx fluid from the parasitoid Campoletis sonorensis

Fifth-instar Heliothis virescens larvae did not pupate after injections of Campoletis sonorensis calyx fluid in or before the burrow-digging stage of development. Arrested development occurred in 40% of larvae injected at the cell-formation stage. Further experiments showed that the particles in calyx fluid were responsible for developmental arrest. Arrested development due to calyx fluid could be reversed by injecting 10 μg of either ecdysone or 20-hydroxyecdysone, although a second injection of 20-hydroxyecdysone was needed for some larvae 3 days after the first treatment. Ecdysteroid production ceased for up to 10 days in 5th-instar H. virescens after calyx-fluid injection. After 10 days, some experimental larvae began to produce ecdysteroids again but remained developmentally arrested. The head, thorax, or abdomen of larvae were isolated by ligations and calyx fluid injected into the isolated body region. After 24 h, ligatures were released and the larvae observed for developmental arrest. Only injections into the isolated thorax stopped development. This, along with ecdysteroid data, indicated that C. sonorensis calyx fluid may directly affect the prothoracic glands of 5th-instar H. virescens.     

General morphology of the brain of the blind cave beetle,Neaphaenops tellkampfii Erichson (Coleoptera : Carabidae)

Neaphaenops tellkampfii (Coleoptera : Carabidae) was collected from Cartmill Cave located in Hart County, Kentucky, U.S.A. This is a cave insect with complete absence of external evidence of eyes or ocelli. The brain of N. tellkampfii has been studied at the light microscope level using Rowell's (1963) silver staining method. Particular attention has been paid to the protocerebrum. One of the notable features of the brain is the dominance of the corpora pedunculata. The corpora pedunculata consists of a calyx, with 3 groups of fibers originating from the Kenyon cells. The stalk is arranged into 2 distinct layers with alpha and beta lobes. The central complex is located anterio-medially beneath the pons cerebralis. It consists of central and ellipsoid bodies and a single ventral tubercle. The ellipsoid body is connected to the beta lobes by a unique chiasmatic fiber tract. The pons cerebralis appears to be formed by 3 distinct groups of globuli cells sending fibers into the pons. The accessory lobes are situated posterio-laterally. The antennal lobes are located posterio-ventrally. Two tubercles were observed lateral to the protocerebrum which may be vestigial optic tubercles. There is no evidence of typical optic lobes or associated fiber tracts. Fiber connections were observed between the calyx and pons cerebralis, the calyx and central body, and also between the calyx and antennal lobe. Two fiber tracts not previously described were observed extending obliquely from the accessory lobe to the beta lobe and protocerebrum.     

Ultrastructure of the corpus luteum of antarctic seals during pregnancy

Summary The fine structure of granulosa lutein cells from three crabeater seals, Lobodon carcinophagus, and two leopard seals, Hydrurga leptonyx, has been studied from early through mid-pregnancy. Analysis of the arrangement and modifications of the cytoplasmic organelles and inclusions has revealed three types of lutein cells throughout the corpus. Type I cell typically possesses a central nucleus and cytoplasm containing very large amounts of smooth and/or fenestrated endoplasmic cisternae which frequently extend from the juxta-nuclear to the periphery of the cell. Type II cell contains a central or eccentric nucleus, moderate amounts of peripheral, smooth and fenestrated cisternae which often form large and concentric membranous whorls, numerous mitochondria and small lipid droplets. Frequently these cells show polarity in the arrangement of the cytoplasmic organelles and inclusions. Type III cell contains predominant large lipid droplets, many mitochondria, and small amounts of smooth and fenestrated cisternae. In light microscopy the type I cell is evenly granular, while the type III cell is highly vacuolated. Type II cells have both granular and vacuolated conditions. Ultrastructural features of type I and II cells suggest that they probably secrete most of the steroids, whereas the primary role of the type III cells appear to be lipid storage.This research was supported by National Science Foundation, Grant No. 1325 from the Office of Antarctic Biology.     

The fine structure of the female reproductive system of Zorotypus caudelli Karny (Zoraptera)

The general structure of the female genital system of Zorotypus caudelli is described. The ovarioles are of the panoistic type. Due to the reduction of the envelope (tunica externa) the ovarioles are in direct contact with the hemolymph like in some other insect groups, Plecoptera included. The calices are much larger in Z. caudelli then in Zorotypus hubbardi and their epithelial cells produce large amounts of secretions, probably protecting the surface of the eggs deposited on the substrate. Eggs taken from the calyx bear a series of long fringes, which are missing in the eggs found in the ovariole, and in other zorapteran species. The long sperm of Z. caudelli and the long spermathecal duct are likely related to a sexual isolating mechanism (cryptic female choice), impeding female re-mating. The apical receptacle and the spermathecal duct - both of ectodermal origin - consist of three cell types. In addition to the cells beneath the cuticle lining the lumen, two other cell types are visible: secretory and canal cells. The cytoplasm of the former is rich in rough endoplasmic reticulum cisterns and Golgi complexes, which produce numerous discrete dense secretory bodies. These products are released into the receiving canal crossing the extracellular cavity of secretory cells, extending over a series of long microvilli. The secretion is transported towards the lumen of the apical receptacle of the spermatheca or to that of the spermathecal duct by a connecting canal formed by the canal cells. It is enriched by material produced by the slender canal cells. Before mating, the sperm cells are enveloped by a thick glycocalyx produced at the level of the male accessory glands, but it is absent when they have reached the apical receptacle, and also in the spermathecal duct lumen. It is likely removed by secretions of the spermatheca. The eggs are fertilized at the level of the common oviduct where the spermathecal duct opens. Two micropyles at the dorsal side of the equator level possibly facilitate fertilization. The presence of these two micropyles is a presumably derived feature shared with Phasmatodea. The fine structure of the female reproductive system of Z. caudelli does not allow to assess the phylogenetic position at the present stage of knowledge. The enlarged calyx and the temporary presence of long fringes on the eggs are potential autapomorphies of Z. caudelli or may indicate relationships with other Zorotypus species.     

A juxtamembrane basolateral targeting motif regulates signaling through a TGF-β pathway receptor in Drosophila

In polarized epithelial cells, receptor–ligand interactions can be restricted by different spatial distributions of the 2 interacting components, giving rise to an underappreciated layer of regulatory complexity. We explored whether such regulation occurs in the Drosophila wing disc, an epithelial tissue featuring the TGF-β family member Decapentaplegic (Dpp) as a morphogen controlling growth and patterning. Dpp protein has been observed in an extracellular gradient within the columnar cell layer of the disc, but also uniformly in the disc lumen, leading to the question of how graded signaling is achieved in the face of 2 distinctly localized ligand pools. We find the Dpp Type II receptor Punt, but not the Type I receptor Tkv, is enriched at the basolateral membrane and depleted at the junctions and apical surface. Wit, a second Type II receptor, shows a markedly different behavior, with the protein detected on all membrane regions but enriched at the apical side. Mutational studies identified a short juxtamembrane sequence required for basolateral restriction of Punt in both wing discs and mammalian Madin-Darby canine kidney (MDCK) cells. This basolateral targeting (BLT) determinant can dominantly confer basolateral localization on an otherwise apical receptor. Rescue of punt mutants with transgenes altered in the targeting motif showed that flies expressing apicalized Punt due to the lack of a functional BLT displayed developmental defects, female sterility, and significant lethality. We also show that apicalized Punt does not produce an ectopic signal, indicating that the apical pool of Dpp is not a significant signaling source even when presented with Punt. Instead, we find that basolateral presentation of Punt is required for optimal signaling. Finally, we present evidence that the BLT acts through polarized sorting machinery that differs between types of epithelia. This suggests a code whereby each epithelial cell type may differentially traffic common receptors to enable distinctive responses to spatially localized pools of extracellular ligands.

Receptor-ligand interactions can be restricted by different spatial distributions of the two interacting components, giving rise to an underappreciated layer of regulatory complexity. This study reveals that an evolutionarily conserved mechanism for polarized delivery of a transmembrane receptor (the Dpp Type II receptor Punt) supports robust morphogen signaling in a model epithelial tissue, the Drosophila wing disc.     

Movement of neurosecretory product through the anatomical compartments of the neural lobe of the pituitary gland

Summary Electron microscopic studies of the carotid body of the domestic fowl (Gallus gallus domesticus) have shown Type I and Type II cells combined with axons into compact groups. The many Type I cells in the depths of the organ had a body, containing the nucleus, and an elongated, flared process. Some of the Type I cells in the superficial regions tended to be spindle-shaped. Type I cells were characterised by membrane-bound, dense-cored vesicles about 120 nm in diameter. Type II cells invested the Type I cells and had axons embedded in them as in Schwann cells.The fine structure of the carotid body in the domestic fowl resembles that of the Lovebird (Uroloncha domestica) and of various amphibia and mammals. The possibility is discussed that the Type I cells may have a chemoreceptor or a general secretory function, or even both pathway for functions together. The main role of the Type II cells seems to be to provide a of these axons leading to or from Type I cells.The authors are grateful to Mr. R. P. Gould of the Department of Anatomy, Middlesex Hospital Medical School for permission to use some of his and Dr. Hodges' original material in the illustrations. Dr. Hodges also wishes to thank the A.R.C. and the University of London Central Research Fund for financial assistance. We are also most appreciative of the photographic assistance of J. Geary.     

The goblet cavity matrix in the larval midgut of Hyalophora cecropia

The larval midgut of the silkmoth Hyalophora cecropia was examined using scanning electron microscopy. Goblet cells were observed to contain within their cavities a matrix plug. This matrix material was extruded onto the lumen side of the epithelium when the tissue was stretched. The rôle of this matrix material in maintenance of the capacity of the midgut to transport ions in vivo and in vitro is discussed.     

Ultrastructural study on cocoon formation in the freshwater oligochaete,Tubifex hattai

The clitellar epithelium of the freshwater oligochaete, Tubifex hattai, is composed of four types of gland cells (Type I, II, III, and IV), in addition to the cells generally found in the epidermis of this worm. The possible function of these gland cells in cocoon formation was studied with the electron microscope. Type I cells discharge their secretory granules by means of compound exocytosis and provide the materials for the future cocoon membrane. Immediately after completion of the discharge from Type I cells, Type II and III cells simultaneously discharge their secretory granules by means of compound exocytosis. The secretions from Type II cells constitute a colloid in the cocoon lumen and probably cause structural modifications in the future cocoon membrane. The secretory products from Type III cells form the cocoon plug. Although the process of discharge of secretory granules from Type IV cells was not observed, the contribution of these cells to the cocoon formation, producing hoops on the outer surface of the future cocoon membrane and fixing its anterior ends on the clitellum, is inferred from a morphological comparison of the hoop and the structure of the secretory granules.     

Effects of calcitonin and parathyroid hormone on the metabolism of chondrocytes in culture

Rabbit articular chondrocytes in suspension culture synthesize Type II colagen [3α₁(II)] in the absence of extracellular Ca²⁺ and Type Icollagen [2α₁?(I)·α₂] in the complete medium. As a result of pre-treatment in monolayer culture with calcitonin or parathyroid hormone in the complete medium, an influx of Ca²⁺ into the cells occurs. These cells produce mainly Type I collagen when transferred to suspension cultures in the medium devoid of CaCl₂. If added directly to the suspension culture medium containing no CaCl₂, calcitonin stimulates an active efflux of Ca²⁺ from the cells into the medium and leads the cells to synthesize Type I collagen. Under similar conditions, parathyroid hormone does not change the collagen-phenotype.     

A rickettsial pathogen of the amphipod Rivulogammarus pulex

A microorganism attributed to the genus Rickettsiella was found as a pathogen of the amphipod Rivulogammarus pulex collected in the south of Sweden. The rickettsiae were studied using light and electron microscopical methods, and different stainings were tested. The polychromatic staining by J. M. Vetterling and D.E. Thompson (1972, Stain Technol., 47, 164–165) appeared most suitable. Several tissues were infected, most heavily in the fat cells. Infection was restricted to the cytoplasm and infected cells were hypertrophied. The rickettsiae developed inside membrane-lined vacuoles and three morphological types were observed. Type 1 was irregular rods with a length of 0.6–1.4 μm; type 2 electron-dense, slightly bent rods of regular shape, 0.5–0.6 μm long; type 3 rounded cells with a diameter of 1.1–2.8 μm containing irregular crystal-like bodies.