scholarly journals Immuno-Ultrastructural Localization and Putative Multiplication Sites of Huanglongbing Bacterium in Asian Citrus Psyllid Diaphorina citri

Insects ◽  
2019 ◽  
Vol 10 (12) ◽  
pp. 422 ◽  
Author(s):  
El-Desouky Ammar ◽  
Diann Achor ◽  
Amit Levy
Keyword(s):  
Salivary Glands ◽  
Close Association ◽  
Ultrastructural Localization ◽  
Ultrastructural Level ◽  
Malpighian Tubules ◽  
Secretory Cells ◽  
Asian Citrus Psyllid ◽  
Bacterial Cells ◽  
Transmission Electron ◽  
Candidatus Liberibacter

Huanglongbing, the most destructive citrus disease worldwide, is caused by the bacterium ‘Candidatus Liberibacter asiaticus’ (CLas) and is vectored by the Asian citrus psyllid (ACP). Very little is known about the form and distribution of CLas in infected psyllids, especially at the ultrastructural level. Here, we examined these aspects by transmission electron microscopy, combined with immunogold labeling. In CLas-exposed ACP adults, the CLas bacterial cells were found to be pleomorphic taking tubular, spherical, or flask-shaped forms, some of which seemed to divide further. Small or large aggregates of CLas were found in vacuolated cytoplasmic pockets of most ACP organs and tissues examined, including the midgut, filter chamber, hindgut, Malpighian tubules, and secretory cells of the salivary glands, in addition to fat tissues, epidermis, muscle, hemocytes, neural tissues, bacteriome, and walls of the female spermatheca and oviduct. Large aggregates of CLas were found outside the midgut within the filter chamber and between the sublayers of the basal lamina of the hindgut and Malpighian tubules. Novel intracytoplasmic structures that we hypothesized as ‘putative CLas multiplication sites’ were found in the cells of the midgut, salivary glands, and other tissues in CLas-exposed ACP. These structures, characterized by containing a granular matrix and closely packed bacterial cells, were unbound by membranes and were frequently associated with rough endoplasmic reticulum. Our results point to the close association between CLas and its psyllid vector, and provide support for a circulative-propagative mode of transmission.

scholarly journals Microscopical characterization of the salivary glands of the carnivorous cephalaspidean Philinopsis depicta (Mollusca, Opisthobranchia)

2009 ◽  
Vol 15 (S3) ◽  
pp. 39-40
Author(s):  
A. Lobo-da-Cunha ◽  
I. Ferreira ◽  
G. Calado
Keyword(s):  
Salivary Glands ◽  
Secretory Cells ◽  
Apical Region ◽  
Ciliated Cells ◽  
Transmission Electron ◽  
Herbivorous Species ◽  
Small Clusters ◽  
The Relationship ◽  
Apical Vacuole

AbstractCephalaspideans are a group of opisthobranch gastropods comprising carnivorous and herbivorous species, allowing an investigation of the relationship between these diets and the morphofunctional features of the salivary glands.In this study, the salivary glands of the carnivorous cephalaspidean Philinopsis depicta were observed by light microscopy using semithin sections and by transmission electron microscopy. A central duct runs along the length of these thin ribbon-shaped glands dividing them in two halves, each formed by a single row of tubules perpendicularly attached to the central duct. The simple epithelium of the central duct and lateral tubes contains ciliated cells and two types of secretory cells, named granular cells and cells with apical vacuole (Fig. 1). A very thin outer layer of connective tissue covers the epithelium (Fig. 1). The ciliated cells are numerous but very thin, forming small clusters between secretory cells. The nucleus, several mitochondria and a few lysosomes are located in the apical region were the cells are wider. A very thin cytoplasmic stalk reaches the base of the epithelium and contains bundles of filaments in addition to some mitochondria.

scholarly journals Early Events of Citrus Greening (Huanglongbing) Disease Development at the Ultrastructural Level

2010 ◽  
Vol 100 (9) ◽  
pp. 949-958 ◽  
Author(s):  
Svetlana Y. Folimonova ◽  
Diann S. Achor
Keyword(s):  
Middle Lamella ◽  
Ultrastructural Level ◽  
Citrus Greening ◽  
Ultrastructural Changes ◽  
Bacterial Cells ◽  
Tissue Samples ◽  
Large Numbers ◽  
Presymptomatic Stage ◽  
Candidatus Liberibacter ◽  
Advanced Stages

Citrus greening (Huanglongbing [HLB]) is one of the most destructive diseases of citrus worldwide. The causal agent of HLB in Florida is thought to be ‘Candidatus Liberibacter asiaticus’. Understanding of the early events in HLB infection is critical for the development of effective measures to control the disease. In this work, we conducted cytopathological studies by following the development of the disease in citrus trees graft inoculated with ‘Ca. L. asiaticus’-containing material under greenhouse conditions to examine the correlation between ultrastructural changes and symptom production, with the main objective of characterizing the early events of infection. Based on our observations, one of the first degenerative changes induced upon invasion of the pathogen appears to be swelling of middle lamella between cell walls surrounding sieve elements. This anatomical aberration was often observed in samples from newly growing flushes in inoculated sweet orange and grapefruit trees at the early “presymptomatic” stage of HLB infection. Development of symptoms and their progression correlated with an increasing degree of microscopic aberrations. Remarkably, the ability to observe the bacterium in the infected tissue also correlated with the degree of the disease progression. Large numbers of bacterial cells were found in phloem sieve tubes in tissue samples from presymptomatic young flushes. In contrast, we did not observe the bacteria in highly symptomatic leaf samples, suggesting a possibility that, at more advanced stages of the disease, a major proportion of ‘Ca. L. asiaticus’ is present in a nonviable state. We trust that observations reported here advance our understanding of how ‘Ca. L. asiaticus’ causes disease. Furthermore, they may be an important aid in answering a question: when and where within an infected tree the tissue serves as a better inoculum source for acquisition and transmission of the bacterium by its psyllid vector.

Cephalic salivary gland ultrastructure of worker and queen eusocial bees (Hymenoptera, Apidae)

2009 ◽  
Vol 59 (3) ◽  
pp. 299-311 ◽  
Author(s):  
Carminda da Cruz-Landim ◽  
Silvana Beani Poiani
Keyword(s):  
Salivary Glands ◽  
Smooth Endoplasmic Reticulum ◽  
Secretory Cells ◽  
Apical Region ◽  
Transmission Electron ◽  
Basal Plasma ◽  
Eusocial Bees ◽  
Scaptotrigona Postica ◽  
Microscopy Techniques

AbstractEusocial bees present a pair of functional salivary glands in head, the cephalic salivary glands. These glands from workers and queens of two eusocial bee species, Apis mellifera and Scaptotrigona postica, were examined at different life stages using routine transmission electron microscopy techniques to correlate morphology and gland functions. Ultrastructural features of worker and queen glands ducts and secretory units were descriptively compared between species. The duct cells present basal plasma membrane invaginations reaching the apical region. Intercellular space and invaginations contain material of similar electron-density to the basal lamina, suggesting that substances might be directly absorbed from the hemolymph to the gland lumen. The secretory cells are rich in smooth endoplasmic reticulum, mitochondria, Golgi, and vesicles typical of lipid secretion. Secretory cells in S. postica become flattened with age in contrast to A. mellifera, where cells remained cuboidal. Mitochondria are associated with secretory vesicles and may become lipid deposits. A possible role of worker and queen secretion is discussed, taking changes in caste gland morphology and their function in the colony into account.

scholarly journals The Anatomy and Ultrastructure of the Digestive Tract and Salivary Glands of Hishimonus lamellatus (Hemiptera: Cicadellidae)

2019 ◽  
Vol 19 (4) ◽  
Author(s):  
Lizhen Dai ◽  
Baodong Yang ◽  
Jinzhong Wang ◽  
Zhiyong Zhang ◽  
Rui Yang ◽  
...  
Keyword(s):  
Salivary Glands ◽  
Digestive Tract ◽  
Secretory Granules ◽  
Malpighian Tubules ◽  
Midgut Epithelium ◽  
Secretory Cells ◽  
Basal Region ◽  
Apical Surface ◽  
Accessory Glands ◽  
Luminal Membrane

AbstractIn recent years, we found that Hishimonus lamellatus Cai et Kuoh is a potential vector of jujube witches’-broom phytoplasma. However, little is known about the anatomy and histology of this leafhopper. Here, we examined histology and ultrastructure of the digestive system of H. lamellatus, both by dissecting and by semi- and ultrathin sectioning techniques. We found that the H. lamellatus digestive tract consists of an esophagus, a filter chamber, a conical midgut and midgut loop, Malpighian tubules, an ileum, and a rectum. Furthermore, both the basal region of the filter chamber epithelium and the apical surface of the midgut epithelium have developed microvilli. We also identify the perimicrovillar membrane, which ensheaths the microvilli of midgut loop enterocyte, and the flame-like luminal membrane, which covers the microvilli of the conical midgut epithelium. In addition, H. lamellatus has the principal and accessory salivary glands. Our observations also showed that the endoplasmic reticulum, mitochondria, and secretory granules were all highly abundant in the secretory cells of the principal salivary glands, while the accessory glands consist of only one ovate or elbow-like acinus. We also briefly contrast the structure of the gut of H. lamellatus with those of other leafhopper species. These results intend to offer help for the future study on the histological and subcellular levels of phytopathogen–leafhopper relationships, including transmission barriers and the binding sites of pathogens and other microorganisms within their leafhopper vectors.

scholarly journals Visualization of ‘Candidatus Liberibacter asiaticus’ Cells in the Vascular Bundle of Citrus Seed Coats with Fluorescence In Situ Hybridization and Transmission Electron Microscopy

2013 ◽  
Vol 103 (6) ◽  
pp. 545-554 ◽  
Author(s):  
Mark E. Hilf ◽  
Kenneth R. Sims ◽  
Svetlana Y. Folimonova ◽  
Diann S. Achor
Keyword(s):  
16S Rrna ◽  
Rrna Gene ◽  
Bacterial Cells ◽  
Candidatus Liberibacter Asiaticus ◽  
Transmission Electron ◽  
Candidatus Liberibacter ◽  
Liberibacter Asiaticus ◽  
Citrus Seed ◽  
Seed Coats

‘Candidatus Liberibacter asiaticus’ is the bacterium implicated as a causal agent of the economically damaging disease of citrus called huanglongbing (HLB). Vertical transmission of the organism through seed to the seedling has not been demonstrated. Previous studies using real-time polymerase chain reaction assays indicated abundant bacterial 16S rRNA sequences in seed coats of citrus seed but the presence of intact bacterial cells was not demonstrated. We used microscopy to verify that intact bacterial cells were present in citrus seed coats. Bacterial cells with the morphology and physical dimensions appropriate for ‘Ca. L. asiaticus’ were seen in phloem sieve elements in the vascular bundle of grapefruit seed coats using transmission electron microscopy (TEM). Fluorescence in situ hybridization (FISH) analyses utilizing probes complementary to the ‘Ca. L. asiaticus’ 16S rRNA gene revealed bacterial cells in the vascular tissue of intact seed coats of grapefruit and pummelo and in fragmented vascular bundles excised from grapefruit seed coats. The physical measurements and the morphology of individual bacterial cells were consistent with those ascribed in the literature to ‘Ca. L. asiaticus’. No bacterial cells were observed in preparations of seed from fruit from noninfected trees. A small library of clones amplified from seed coats from a noninfected tree using degenerate primers targeting prokaryote 16S rRNA gene sequences contained no ‘Ca. L. asiaticus’ sequences, whereas 95% of the sequences in a similar library from DNA from seed coats from an infected tree were identified as ‘Ca. L. asiaticus’, providing molecular genetic corroboration that the bacterial cells observed by TEM and FISH in seed coats from infected trees were ‘Ca. L. asiaticus’.

The energy filtering TEM (EFTEM) in modern biological transmisson Electron Microscopy

1995 ◽  
Vol 53 ◽  
pp. 668-669
Author(s):  
W. Probst ◽  
V.E. Bayer
Keyword(s):  
Electron Microscopy ◽  
Chemical Elements ◽  
Ultrastructural Localization ◽  
Ultrastructural Level ◽  
Limited Information ◽  
Energy Filtering ◽  
Transmission Electron ◽  
New Findings ◽  
Biochemical Analyses ◽  
Selection Of

Modern biological electron microscopy can no longer be a static tool merely describing morphology. In addition to ultrastructural information, insights into the molecular and chemical composition of a sample are needed so that new findings stemming from molecular biological and biochemical analyses can be given meaning in an ultrastructural context. Biological electron microscopy will be an essential tool for future discoveries involving the ultrastructural localization of molecules and chemical elements, and it will provide a means to identify the ultrastructural basis for a variety of reaction mechanisms. Many messenger compounds are currently known which can produce dynamic changes of either a subtle or dramatic nature at the ultrastructural level, but only the most basic of these can be examined using a conventional transmission electron microscope (CTEM). CTEMs provide limited information because they perform conventional imaging and do not employ all the signals available for analysis. Unlike a CTEM, an EFTEM permits the selection of a defined energy (wavelength) of electrons which are then used for imaging.

scholarly journals Colonization of Dodder, Cuscuta indecora, by ‘Candidatus Liberibacter asiaticus’ and ‘Ca. L. americanus’

2010 ◽  
Vol 100 (8) ◽  
pp. 756-762 ◽  
Author(s):  
John S. Hartung ◽  
Cristina Paul ◽  
Diann Achor ◽  
R. H. Brlansky
Keyword(s):  
Cross Sections ◽  
Quantitative Polymerase Chain Reaction ◽  
Morphological Differentiation ◽  
Ultrastructural Level ◽  
Bacterial Cells ◽  
Candidatus Liberibacter Asiaticus ◽  
Citrus Industry ◽  
Vessel Elements ◽  
Candidatus Liberibacter ◽  
Liberibacter Asiaticus

Huanglongbing, or citrus greening, threatens the global citrus industry. The presumptive pathogens, ‘Candidatus Liberibacter asiaticus’ and ‘Ca. L. americanus’ can be transferred from citrus to more easily studied experimental hosts by using holoparasitic dodder plants. However, the interaction between ‘Candidatus Liberibacter’ spp. and the dodder has not been studied. We combined quantitative polymerase chain reaction with electron microscopy to show that only 65% of tendrils of Cuscuta indecora grown on ‘Ca. Liberibacter’ spp.-infected host plants had detectable levels of the pathogen. Among tendrils that were colonized by Liberibacter in at least one 2 cm segment, most were not colonized in all segments. Furthermore, the estimated population levels of the pathogen present in serial 2 cm segments of dodder tendrils varied widely and without any consistent pattern. Thus, there was generally not a concentration gradient of the pathogen from the source plant towards the recipient and populations of the pathogen were sometimes found in the distal segments of the dodder plant but not in the proximal or middle segments. Populations of the pathogens ranged from 2 × 102 to 3.0 × 108 cells per 2 cm segment. On a fresh weight basis, populations as high as 1.4 × 1010 cells per g of tissue were observed demonstrating that ‘Ca. Liberibacter’ spp. multiplies well in Cuscuta indecora. However, 55% of individual stem segments did not contain detectable levels of the pathogen, consistent with a pattern of nonuniform colonization similar to that observed in the much more anatomically complex citrus tree. Colonization of dodder by the pathogen is also nonuniform at the ultrastructural level, with adjacent phloem vessel elements being completely full of the pathogen or free of the pathogen. We also observed bacteria in the phloem vessels that belonged to two distinct size classes based on the diameters of cross sections of cells. In other sections from the same tendrils we observed single bacterial cells that were apparently in the process of differentiating between the large and round forms to the long and thin forms (or vice versa). The process controlling this morphological differentiation of the pathogen is not known. The highly reduced and simplified anatomy of the dodder plant as well as its rapid growth rate compared with citrus, and the ability of the plant to support multiplication of the pathogen to high levels, makes it an interesting host plant for further studies of host–pathogen interactions.

Ultrastructural Analysis of the Salivary Glands of Gromphadorhina Portentosa (Schaum)

1977 ◽  
Vol 35 ◽  
pp. 638-639
Author(s):  
P.J. Dailey
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Salivary Glands ◽  
Secretory Vesicles ◽  
The Past ◽  
Transmission Electron ◽  
Means Of Transmission ◽  
Gromphadorhina Portentosa ◽  
Scanning Electron ◽  
Topographical Relief

The structure of insect salivary glands has been extensively investigated during the past decade; however, none have attempted scanning electron microscopy (SEM) in ultrastructural examinations of these secretory organs. This study correlates fine structure by means of SEM cryofractography with that of thin-sectioned epoxy embedded material observed by means of transmission electron microscopy (TEM).Salivary glands of Gromphadorhina portentosa were excised and immediately submerged in cold (4°C) paraformaldehyde-glutaraldehyde fixative1 for 2 hr, washed and post-fixed in 1 per cent 0s04 in phosphosphate buffer (4°C for 2 hr). After ethanolic dehydration half of the samples were embedded in Epon 812 for TEM and half cryofractured and subsequently critical point dried for SEM. Dried specimens were mounted on aluminum stubs and coated with approximately 150 Å of gold in a cold sputtering apparatus.Figure 1 shows a cryofractured plane through a salivary acinus revealing topographical relief of secretory vesicles.

Scanning and Transmission Electron Microscopy of the Endometrium in Women with Ovarian Dysgenesis (Turner'S Syndrome)

1976 ◽  
Vol 34 ◽  
pp. 148-149
Author(s):  
A. González-Angulo ◽  
S. Armendares-Sagrera ◽  
I. Ruíz de Chávez ◽  
H. Marquez-Monter ◽  
R. Aznar
Keyword(s):  
Surface Epithelium ◽  
Secretory Cells ◽  
Turner's Syndrome ◽  
Ciliated Cells ◽  
Turner’S Syndrome ◽  
Exogenous Hormone ◽  
Ovarian Dysgenesis ◽  
Transmission Electron ◽  
Normal Women ◽  
Microscopy Examination

It is a well documented fact that endometrial hyperplasia and adenocarcinoma may develop in women with Turner's syndrome who had received unopposed estrogen treatment (1), as well as in normal women under contraceptive medication with the sequential regime (2). The purpose of the present study was to characterize the possible changes in surface and glandular epithelium in these women who were treated with a sequential regime for a period of between three and eight years. The aim was to find organelle modifications which may lead to the understanding of the biology of an endometrium under exogenous hormone stimulation. Light microscopy examination of endometrial biopsies of nine patients disclosed a proliferative pattern; in two of these, there was focal hyperplasia. With the scanning electron microscope the surface epithelium in all biopsies showed secretory cells with microvilli alternating with non secretory ciliated cells. Regardless of the day of the cycle all biopsies disclosed a large number of secretory cells rich in microvilli (fig.l) with long and slender projections some of which were branching (fig. 2).

Ultrastructural Localization of Antigens by Capillary Action Immunocytochemistry

1996 ◽  
Vol 54 ◽  
pp. 40-41
Author(s):  
László G. Kömüves
Keyword(s):  
San Francisco ◽  
Colloidal Gold ◽  
Specific Binding ◽  
Ultrastructural Localization ◽  
Ultrastructural Level ◽  
Uranyl Acetate ◽  
Adhesive Tape ◽  
Distilled Water ◽  
Capillary Action ◽  
Rabbit Igg

Light microscopic immunohistochemistry based on the principle of capillary action staining is a widely used method to localize antigens. Capillary action immunostaining, however, has not been tested or applied to detect antigens at the ultrastructural level. The aim of this work was to establish a capillary action staining method for localization of intracellular antigens, using colloidal gold probes.Post-embedding capillary action immunocytochemistry was used to detect maternal IgG in the small intestine of newborn suckling piglets. Pieces of the jejunum of newborn piglets suckled for 12 h were fixed and embedded into LR White resin. Sections on nickel grids were secured on a capillary action glass slide (100 μm wide capillary gap, Bio-Tek Solutions, Santa Barbara CA, distributed by CMS, Houston, TX) by double sided adhesive tape. Immunolabeling was performed by applying reagents over the grids using capillary action and removing reagents by blotting on filter paper. Reagents for capillary action staining were from Biomeda (Foster City, CA). The following steps were performed: 1) wet the surface of the sections with automation buffer twice, 5 min each; 2) block non-specific binding sites with tissue conditioner, 10 min; 3) apply first antibody (affinity-purified rabbit anti-porcine IgG, Sigma Chem. Co., St. Louis, MO), diluted in probe diluent, 1 hour; 4) wash with automation buffer three times, 5 min each; 5) apply gold probe (goat anti-rabbit IgG conjugated to 10 nm colloidal gold, Zymed Laboratories, South San Francisco, CA) diluted in probe diluent, 30 min; 6) wash with automation buffer three times, 5 min each; 7) post-fix with 5% glutaraldehyde in PBS for 10 min; 8) wash with PBS twice, 5 min each; 9) contrast with 1% OSO4 in PBS for 15 min; 10) wash with PBS followed by distilled water for5 min each; 11) stain with 2% uranyl acetate for 10 min; 12) stain with lead citrate for 2 min; 13) wash with distilled water three times, 1 min each. The glass slides were separated, and the grids were air-dried, then removed from the adhesive tape. The following controls were used to ensure the specificity of labeling: i) omission of the first antibody; ii) normal rabbit IgG in lieu of first antibody; iii) rabbit anti-porcine IgG absorbed with porcine IgG.

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