scholarly journals Extracellular excystation and development of Cryptosporidium: tracing the fate of oocysts within Pseudomonas aquatic biofilm systems

2021 ◽  
Author(s):  
Wan Koh ◽  
Andrew Thompson ◽  
Hanna Edwards ◽  
Paul Monis ◽  
Peta L Clode
Keyword(s):  
Laser Scanning ◽  
Distribution Systems ◽  
Water Distribution ◽  
Developmental Stages ◽  
Parasitophorous Vacuole ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Growth Environment ◽  
Flow Cytometric ◽  
Harsh Conditions

Background Aquatic biofilms often serve as environmental reservoirs for microorganisms and provide them with a nutrient-rich growth environment under harsh conditions. With regard to Cryptosporidium, biofilms can serve as environmental reservoirs for oocysts, but may also support the growth of additional Cryptosporidium stages. Results Here we used confocal laser scanning microscopy, scanning electron microscopy (SEM), and flow cytometry to identify and describe various Cryptosporidium developmental stages present within aquatic biofilm systems, and to directly compare these to stages produced in cell culture. We also show that Cryptosporidium has the ability to form a parasitophorous vacuole independently, in a host-free biofilm environment, potentially allowing them to complete an extracellular life cycle. Correlative data from confocal and SEM imaging of the same cells confirmed that the observed developmental stages (including trophozoites, meronts, and merozoites) were Cryptosporidium. These microscopy observations were further supported by flow cytometric analyses, where excysted oocyst populations were detected in 1, 3 and 6 day-old Cryptosporidium-exposed biofilms, but not in biofilm-free controls. Conclusions These observations not only highlight the risk that aquatic biofilms pose in regards to Cryptosporidium outbreaks from water distribution systems, but further indicate that even simple biofilms are able to stimulate oocyst excystation and support the extracellular multiplication and development of Cryptosporidium within aquatic environments.

scholarly journals Extracellular excystation and development of Cryptosporidium: tracing the fate of oocysts within Pseudomonas aquatic biofilm systems

2021 ◽  
Author(s):  
Wan Koh ◽  
Andrew Thompson ◽  
Hanna Edwards ◽  
Paul Monis ◽  
Peta L Clode
Keyword(s):  
Laser Scanning ◽  
Distribution Systems ◽  
Water Distribution ◽  
Developmental Stages ◽  
Parasitophorous Vacuole ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Growth Environment ◽  
Flow Cytometric ◽  
Harsh Conditions

Background Aquatic biofilms often serve as environmental reservoirs for microorganisms and provide them with a nutrient-rich growth environment under harsh conditions. With regard to Cryptosporidium, biofilms can serve as environmental reservoirs for oocysts, but may also support the growth of additional Cryptosporidium stages. Results Here we used confocal laser scanning microscopy, scanning electron microscopy (SEM), and flow cytometry to identify and describe various Cryptosporidium developmental stages present within aquatic biofilm systems, and to directly compare these to stages produced in cell culture. We also show that Cryptosporidium has the ability to form a parasitophorous vacuole independently, in a host-free biofilm environment, potentially allowing them to complete an extracellular life cycle. Correlative data from confocal and SEM imaging of the same cells confirmed that the observed developmental stages (including trophozoites, meronts, and merozoites) were Cryptosporidium. These microscopy observations were further supported by flow cytometric analyses, where excysted oocyst populations were detected in 1, 3 and 6 day-old Cryptosporidium-exposed biofilms, but not in biofilm-free controls. Conclusions These observations not only highlight the risk that aquatic biofilms pose in regards to Cryptosporidium outbreaks from water distribution systems, but further indicate that even simple biofilms are able to stimulate oocyst excystation and support the extracellular multiplication and development of Cryptosporidium within aquatic environments.

scholarly journals Protocol for rapid clearing and staining of fixed Arabidopsis ovules for improved imaging by confocal laser scanning microscopy

Plant Methods ◽  
2019 ◽  
Vol 15 (1) ◽  
Author(s):  
Rachele Tofanelli ◽  
Athul Vijayan ◽  
Sebastian Scholz ◽  
Kay Schneitz
Keyword(s):  
Laser Scanning ◽  
Developmental Stages ◽  
Imaging Techniques ◽  
Three Dimensional ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Imaging Data ◽  
Model Species ◽  
Cellular Models ◽  
Cellular Resolution

Abstract Background A salient topic in developmental biology relates to the molecular and genetic mechanisms that underlie tissue morphogenesis. Modern quantitative approaches to this central question frequently involve digital cellular models of the organ or tissue under study. The ovules of the model species Arabidopsis thaliana have long been established as a model system for the study of organogenesis in plants. While ovule development in Arabidopsis can be followed by a variety of different imaging techniques, no experimental strategy presently exists that enables an easy and straightforward investigation of the morphology of internal tissues of the ovule with cellular resolution. Results We developed a protocol for rapid and robust confocal microscopy of fixed Arabidopsis ovules of all stages. The method combines clearing of fixed ovules in ClearSee solution with marking the cell outline using the cell wall stain SCRI Renaissance 2200 and the nuclei with the stain TO-PRO-3 iodide. We further improved the microscopy by employing a homogenous immersion system aimed at minimizing refractive index differences. The method allows complete inspection of the cellular architecture even deep within the ovule. Using the new protocol we were able to generate digital three-dimensional models of ovules of various stages. Conclusions The protocol enables the quick and reproducible imaging of fixed Arabidopsis ovules of all developmental stages. From the imaging data three-dimensional digital ovule models with cellular resolution can be rapidly generated using image analysis software, for example MorphographX. Such digital models will provide the foundation for a future quantitative analysis of ovule morphogenesis in a model species.

scholarly journals Ovule Development and in Planta Transformation of Paphiopedilum Maudiae by Agrobacterium-Mediated Ovary-Injection

2020 ◽  
Vol 22 (1) ◽  
pp. 84
Author(s):  
Bai-Xue Luo ◽  
Li Zhang ◽  
Feng Zheng ◽  
Kun-Lin Wu ◽  
Lin Li ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Developmental Stages ◽  
Embryo Sac ◽  
Mature Embryo ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Pcr Analysis ◽  
In Planta Transformation ◽  
In Planta ◽  
Hygromycin Selection

In this paper, the development of the Paphiopedilum Maudiae embryo sac at different developmental stages after pollination was assessed by confocal laser scanning microscopy. The mature seeds of P. Maudiae consisted of an exopleura and a spherical embryo, but without an endosperm, while the inner integument cells were absorbed by the developing embryo. The P. Maudiae embryo sac exhibited an Allium type of development. The time taken for the embryo to develop to a mature sac was 45-50 days after pollination (DAP) and most mature embryo sacs had completed fertilization and formed zygotes by about 50–54 DAP. In planta transformation was achieved by injection of the ovaries by Agrobacterium, resulting in 38 protocorms or seedlings after several rounds of hygromycin selection, corresponding to 2, 7, 5, 1, 3, 4, 9, and 7 plantlets from Agrobacterium-mediated ovary-injection at 30, 35, 42, 43, 45, 48, 50, and 53 DAP, respectively. Transformation efficiency was highest at 50 DAP (2.54%), followed by 2.48% at 53 DAP and 2.45% at 48 DAP. Four randomly selected hygromycin-resistant plants were GUS-positive after PCR analysis. Semi-quantitative PCR and quantitative real-time PCR analysis revealed the expression of the hpt gene in the leaves of eight hygromycin-resistant seedlings following Agrobacterium-mediated ovary-injection at 30, 35, 42, 43, 45, 48, 50, and 53 DAP, while hpt expression was not detected in the control. The best time to inject P. Maudiae ovaries in planta with Agrobacterium is 48-53 DAP, which corresponds to the period of fertilization. This protocol represents the first genetic transformation protocol for any Paphiopedilum species and will allow for expanded molecular breeding programs to introduce useful and interesting genes that can expand its ornamental and horticulturally important characteristics.

scholarly journals From swimming towards sessility in two metamorphoses – the drastic changes in structure and function of the nervous system of the bay barnacle Amphibalanus improvisus (Crustacea, Thecostraca, Cirripedia) during development

2020 ◽  
Vol 89 (3) ◽  
pp. 324-352
Author(s):  
Paul Kalke ◽  
Thomas Frase ◽  
Stefan Richter
Keyword(s):  
Nervous System ◽  
Peripheral Nervous System ◽  
Nerve Cord ◽  
Laser Scanning ◽  
Ventral Nerve Cord ◽  
Developmental Stages ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Compound Eyes ◽  
Cephalic Shield

Knowledge about the development of the nervous system in cirripeds is limited, particularly with regard to the changes that take place during the two metamorphoses their larvae undergo. This study delivers the first detailed description of the development of the nervous system in a cirriped species, Amphibalanus improvisus by using immunohistochemical labeling against acetylated alpha-tubulin, and confocal laser scanning microscopy. The development of the nervous system in the naupliar stages corresponds largely to that in other crustaceans. As development progresses, the protocerebral sensory organs differentiate and the intersegmental nerves forming the complex peripheral nervous system appear, innervating the sensory structures of the cephalic shield. During metamorphosis into a cypris the lateral sides of the cephalic shield fold down into a bilateral carapace, which leads to a reorganization of the peripheral nervous system. The syncerebrum of the cypris exhibits the highest degree of complexity of all developmental stages, innervating the frontal filaments, nauplius eye, compound eyes and the antennules. During settlement, when the second metamorphosis occur, the closely associated frontal filaments and compound eyes are shed together with the cuticle of the carapace and the antennules. In adults, the syncerebral structures are reduced while the ventral nerve cord and the peripheral nervous system increase in complexity. The peripheral nervous system plays an important role in processing sensory input and also in settlement. In summary, through the larval development we observed a structural and thus also functional increase of complexity in favor of the peripheral nervous system and the ventral nerve cord.

The development of exo-erythrocytic schizonts ofPlasmodium berghei in vitrofrom gamma-irradiated and non-irradiated sporozoites: a study using confocal laser scanning microscopy

Parasitology ◽  
1991 ◽  
Vol 103 (1) ◽  
pp. 17-21 ◽  
Author(s):  
R. E. Sinden ◽  
A. Couchman ◽  
A. Suhrbier ◽  
F. Marsh ◽  
L. Winger ◽  
...  
Keyword(s):  
Laser Scanning ◽  
De Novo ◽  
Parasitophorous Vacuole ◽  
Hepatoma Cell ◽  
Specific Antigen ◽  
Antigen Expression ◽  
Laser Scanning Microscopy ◽  
Host Cells ◽  
Confocal Scanning Laser Microscopy ◽  
Confocal Laser

Confocal scanning laser microscopy has been used to study the distribution of antigens expressed by the liver stages ofPlasmodium bergheiin cultured hepatoma cells. The 3-dimensional images obtained of intact parasites clearly show complex patterns of antigen expression not apparent when using conventional IFAT or immunoelectron microscopy. A liver-stage specific antigen (Pbl 1) was shown to be confined to the parasitophorous vacuole; the vacuole has extensive diverticulae extending into the host cell. Small parasites were detected for the first time in ‘mature’ cultures. These did not represent a distinct population, but the ‘tail’ of a broad continuum of parasite sizes. Irradiated sporozoites produce a transient population of slow-growing parasites which express a very limited range of antigensde novoin the invaded hepatoma cell. A comparison of the reactivity of text-abstract EE parasites with anti-circumsporozoite antibody and with anti-Pbl 1 suggests that the former reagent may reliably be used to identify sporozoites invading host cells, but should not be used to determine the number of parasites that successfully undergo intrahepatic development. Anti-Pbl-1 indicates on 33% of invaded sporozoites identified by anti-CSP subsequently differentiate.

scholarly journals Developmental Table and Three-Dimensional Embryological Image Resource of the Ascidian Ascidiella aspersa

2021 ◽  
Vol 9 ◽  
Author(s):  
Haruka M. Funakoshi ◽  
Takumi T. Shito ◽  
Kotaro Oka ◽  
Kohji Hotta
Keyword(s):  
Laser Scanning ◽  
Developmental Stages ◽  
Model Organism ◽  
Tail Length ◽  
Developmental Time ◽  
Laser Scanning Microscopy ◽  
System Level ◽  
Confocal Laser ◽  
Cross Sectional ◽  
Ascidiella Aspersa

Ascidiella aspersa is an ascidian in the class of chordates—the closest relatives of vertebrates. A. aspersa is a potential model organism for bio-imaging studies due to its extremely transparent embryos as well as is a globally distributed cosmopolitan species. However, there is no standard developmental table for this organism. Here, as a first step to establish A. aspersa as a model organism, we report a standard developmental table as a web-based digital image resource. This resource used confocal laser scanning microscopy to scan more than 3,000 cross-sectional images and 3D-reconstructed images of A. aspersa embryos during embryogenesis. With reference to the standardized developmental table of Ciona intestinalis type A, 26 different developmental stages (Stages 1–26) from fertilized eggs to hatched larvae were redefined for A. aspersa. Cell lineages up to the cleavage period were annotated: The cleavage patterns, the embryonic morphology, and the developmental time were then compared with Ciona. We found that the cleavage patterns and developmental time up to the neurula period in A. aspersa were extremely conserved versus. Ciona. The ratio of the trunk and tail length in the tailbud period were smaller than Ciona indicating a relatively short tail. In addition, the timing of the bending of the tail is earlier than Ciona. This A. aspersa standard 3D digital resource is essential for connecting different omics data to different spatiotemporal hierarchies and is useful for a system-level understanding of chordate development and evolution.

scholarly journals Atypical EnteropathogenicEscherichia coliStrains form Biofilm on Abiotic Surfaces Regardless of Their Adherence Pattern on Cultured Epithelial Cells

2014 ◽  
Vol 2014 ◽  
pp. 1-10 ◽  
Author(s):  
Hebert F. Culler ◽  
Cristiane M. Mota ◽  
Cecilia M. Abe ◽  
Waldir P. Elias ◽  
Marcelo P. Sircili ◽  
...  
Keyword(s):  
Biofilm Formation ◽  
Laser Scanning ◽  
Developmental Stages ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Abiotic Surface ◽  
Abiotic Surfaces ◽  
Significant Difference ◽  
Cultured Epithelial Cells ◽  
Adherence Pattern

The aim of this study was to determine the capacity of biofilm formation of atypical enteropathogenicEscherichia coli(aEPEC) strains on abiotic and biotic surfaces. Ninety-one aEPEC strains, isolated from feces of children with diarrhea, were analyzed by the crystal violet (CV) assay on an abiotic surface after 24 h of incubation. aEPEC strains representing each HEp-2 cell type of adherence were analyzed after 24 h and 6, 12, and 18 days of incubation at 37°C on abiotic and cell surfaces by CFU/cm2counting and confocal laser scanning microscopy (CLSM). Biofilm formation on abiotic surfaces occurred in 55 (60.4%) of the aEPEC strains. There was no significant difference in biofilm biomass formation on an abiotic versus prefixed cell surface. The biofilms could be visualized by CLSM at various developmental stages. aEPEC strains are able to form biofilm on an abiotic surface with no association with their adherence pattern on HEp-2 cells with the exception of the strains expressing UND (undetermined adherence). This study revealed the capacity of adhesion and biofilm formation by aEPEC strains on abiotic and biotic surfaces, possibly playing a role in pathogenesis, mainly in cases of persistent diarrhea.

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