scholarly journals Improved ultrastructure of marine invertebrates using non-toxic buffers

PeerJ ◽  
2016 ◽  
Vol 4 ◽  
pp. e1860 ◽  
Author(s):  
Jacqueline Montanaro ◽  
Daniela Gruber ◽  
Nikolaus Leisch
Keyword(s):  
High Pressure ◽  
Marine Biology ◽  
Marine Invertebrates ◽  
Marine Invertebrate ◽  
Gill Tissue ◽  
Field Work ◽  
High Pressure Freezing ◽  
Invertebrate Species ◽  
Sampling Locations ◽  
Ultrastructure Preservation

Many marine biology studies depend on field work on ships or remote sampling locations where sophisticated sample preservation techniques (e.g., high-pressure freezing) are often limited or unavailable. Our aim was to optimize the ultrastructural preservation of marine invertebrates, especially when working in the field. To achieve chemically-fixed material of the highest quality, we compared the resulting ultrastructure of gill tissue of the musselMytilus eduliswhen fixed with differently buffered EM fixatives for marine specimens (seawater, cacodylate and phosphate buffer) and a new fixative formulation with the non-toxic PHEM buffer (PIPES, HEPES, EGTA and MgCl2). All buffers were adapted for immersion fixation to form an isotonic fixative in combination with 2.5% glutaraldehyde. We showed that PHEM buffer based fixatives resulted in equal or better ultrastructure preservation when directly compared to routine standard fixatives. These results were also reproducible when extending the PHEM buffered fixative to the fixation of additional different marine invertebrate species, which also displayed excellent ultrastructural detail. We highly recommend the usage of PHEM-buffered fixation for the fixation of marine invertebrates.

Trace Metal Accumulation in Marine Invertebrates: Marine Biology or Marine Chemistry?

1997 ◽  
Vol 77 (1) ◽  
pp. 195-210 ◽  
Author(s):  
Philip S. Rainbow
Keyword(s):  
Trace Metal ◽  
Marine Biology ◽  
Metal Accumulation ◽  
Marine Invertebrates ◽  
Sea Water ◽  
Short Review ◽  
Equivalent Weight ◽  
Closely Related Species ◽  
Metal Chemistry ◽  
Invertebrate Species

Trace metals are accumulated by marine invertebrates to body concentrations higher, in many cases orders of magnitude higher, than the concentrations in an equivalent weight of the surrounding sea-water (Eisler, 1981; Rainbow, 1990; Phillips & Rainbow, 1993). Specific details of trace metal accumulation processes vary within the same invertebrate species between metals, and for the same trace metal between invertebrates, often between closely related species (Rainbow, 1990, 1993). This short review attempts to highlight some of the comparative aspects of the processes involved that are expected and explicable in terms of the chemistry of the respective elements, and those where the physiology of the species involved intervenes to offset predictions from purely chemical principles. Although an appreciation of trace metal chemistry is crucial to an understanding of trace metal accumulation, idiosyncrasies in the biology of the invertebrate (at any taxon level) may intervene to bring about significant and unexpected comparative differences in metal accumulation patterns.

scholarly journals Cartilage ultrastructure after high pressure freezing, freeze substitution, and low temperature embedding. II. Intercellular matrix ultrastructure - preservation of proteoglycans in their native state.

1984 ◽  
Vol 98 (1) ◽  
pp. 277-282 ◽  
Author(s):  
E B Hunziker ◽  
R K Schenk
Keyword(s):  
High Pressure ◽  
Low Temperature ◽  
Freeze Substitution ◽  
Epiphyseal Cartilage ◽  
Cartilage Tissue ◽  
High Pressure Freezing ◽  
Intercellular Matrix ◽  
The Matrix ◽  
Protein Components ◽  
Ultrastructure Preservation

The extracellular matrix of epiphyseal cartilage tissue was preserved in a state believed to resemble closely that of native tissue following processing by high pressure freezing, freeze substitution, and low temperature embedding (HPF/FS). Proteoglycans (PG) were preserved in an extended state and were apparent as a reticulum of fine filamentous threads throughout the matrix. Within this network, two morphologically discrete components were discernible and identified with the carbohydrate and protein components of PG molecules. Numerous points of contact were clearly visible between components of the PG network and cross-sectioned collagen fibrils and also between PG components and chondrocytic plasmalemmata. These observations provide direct morphological indication that such relationships may exist in native epiphyseal cartilage tissue.

scholarly journals Effects of temperature on heat-shock responses and survival of two species of marine invertebrates from sub-Antarctic Marion Island

2013 ◽  
Vol 26 (2) ◽  
pp. 145-152 ◽  
Author(s):  
S. Clusella-Trullas ◽  
L. Boardman ◽  
K.T. Faulkner ◽  
L.S. Peck ◽  
S.L. Chown
Keyword(s):  
High Temperature ◽  
Heat Shock ◽  
Heat Shock Response ◽  
Marine Invertebrates ◽  
Marine Invertebrate ◽  
Marion Island ◽  
Cellular Mechanisms ◽  
Invertebrate Species ◽  
Shock Response ◽  
Heat Shock Responses

AbstractThis study examined high temperature survival and heat shock protein 70 (Hsp70) responses to temperature variation for two marine invertebrate species on sub-Antarctic Marion Island. The isopod Exosphaeroma gigas Leach and the amphipod Hyale hirtipalma Dana had the same tolerance to high temperature. The mean upper temperature which was lethal for 50% of the population (upper lethal temperature, ULT50) was 26.4°C for both species. However, the isopod E. gigas showed significant plasticity of ULT50, with a positive response to acclimation. In addition, the isopod had a heat shock response of Hsp70 at all acclimations, and the amount of Hsp70 protein increased significantly from basal levels upon an acute warm exposure after a cold acclimation. By contrast, the amphipod H. hirtipalma showed limited plasticity of ULT50 and no evidence for a heat shock response (failure of three different Hsp70 antibodies to bind to the extracted 70kDa proteins). Overall, these results reflect different flexibility of thermal tolerance of intertidal invertebrate species on Marion Island, with possible variation in the underlying cellular mechanisms, suggesting that warming associated with climate change may result in changes in species assemblage structure in sub-polar environments.

scholarly journals Biochemical evidences for M1-, M17- and M18-like aminopeptidases in marine invertebrates from Cuban coastline

2020 ◽  
Vol 75 (11-12) ◽  
pp. 397-407
Author(s):  
Isel Pascual Alonso ◽  
Laura Rivera Méndez ◽  
Mario E. Valdés-Tresanco ◽  
Lotfi Bounaadja ◽  
Marjorie Schmitt ◽  
...  
Keyword(s):  
Active Site ◽  
Marine Invertebrates ◽  
Marine Invertebrate ◽  
Biochemical Properties ◽  
Peptide Substrates ◽  
Invertebrate Species ◽  
Physiological Processes ◽  
N Terminus ◽  
Optimal Ph

AbstractMetallo-aminopeptidases (mAPs) control many physiological processes. They are classified in different families according to structural similarities. Neutral mAPs catalyze the cleavage of neutral amino acids from the N-terminus of proteins or peptide substrates; they need one or two metallic cofactors in their active site. Information about marine invertebrate’s neutral mAPs properties is scarce; available data are mainly derived from genomics and cDNA studies. The goal of this work was to characterize the biochemical properties of the neutral APs activities in eight Cuban marine invertebrate species from the Phyla Mollusca, Porifera, Echinodermata, and Cnidaria. Determination of substrate specificity, optimal pH and effects of inhibitors (1,10-phenanthroline, amastatin, and bestatin) and cobalt on activity led to the identification of distinct neutral AP-like activities, whose biochemical behaviors were similar to those of the M1 and M17 families of mAPs. Additionally, M18-like glutamyl AP activities were detected. Thus, marine invertebrates express biochemical activities likely belonging to various families of metallo-aminopeptidases.

High-pressure freezing of cells in suspension for low-voltage scanning electron microscopy (LVSEM)

1991 ◽  
Vol 49 ◽  
pp. 64-65
Author(s):  
Marek Malecki ◽  
James Pawley ◽  
Hans Ris
Keyword(s):  
Electron Microscopy ◽  
High Pressure ◽  
Low Voltage ◽  
Culture Media ◽  
Cell Structure ◽  
Freeze Substitution ◽  
Ice Crystal ◽  
High Pressure Freezing ◽  
Cell Culture Media ◽  
Voltage Scanning

The ultrastructure of cells suspended in physiological fluids or cell culture media can only be studied if the living processes are stopped while the cells remain in suspension. Attachment of living cells to carrier surfaces to facilitate further processing for electron microscopy produces a rapid reorganization of cell structure eradicating most traces of the structures present when the cells were in suspension. The structure of cells in suspension can be immobilized by either chemical fixation or, much faster, by rapid freezing (cryo-immobilization). The fixation speed is particularly important in studies of cell surface reorganization over time. High pressure freezing provides conditions where specimens up to 500μm thick can be frozen in milliseconds without ice crystal damage. This volume is sufficient for cells to remain in suspension until frozen. However, special procedures are needed to assure that the unattached cells are not lost during subsequent processing for LVSEM or HVEM using freeze-substitution or freeze drying. We recently developed such a procedure.

Cryosectioning plant roots prepared by high-pressure freezing

1987 ◽  
Vol 45 ◽  
pp. 662-663
Author(s):  
R.E. Crang ◽  
M. Mueller ◽  
K. Zierold
Keyword(s):  
High Pressure ◽  
Cell Walls ◽  
Plant Tissues ◽  
Ice Crystal ◽  
High Pressure Freezing ◽  
Vitreous State ◽  
Radial Depth ◽  
Irregular Topography ◽  
Considerable Depth ◽  
Conventional Freezing

Obtaining frozen-hydrated sections of plant tissues for electron microscopy and microanalysis has been considered difficult, if not impossible, due primarily to the considerable depth of effective freezing in the tissues which would be required. The greatest depth of vitreous freezing is generally considered to be only 15-20 μm in animal specimens. Plant cells are often much larger in diameter and, if several cells are required to be intact, ice crystal damage can be expected to be so severe as to prevent successful cryoultramicrotomy. The very nature of cell walls, intercellular air spaces, irregular topography, and large vacuoles often make it impractical to use immersion, metal-mirror, or jet freezing techniques for botanical material.However, it has been proposed that high-pressure freezing (HPF) may offer an alternative to the more conventional freezing techniques, inasmuch as non-cryoprotected specimens may be frozen in a vitreous, or near-vitreous state, to a radial depth of at least 0.5 mm.

High-pressure freezing and freeze substitution of plant tissue

1992 ◽  
Vol 50 (1) ◽  
pp. 748-749
Author(s):  
William P. Sharp ◽  
Robert W. Roberson
Keyword(s):  
High Pressure ◽  
Cell Structure ◽  
Leaf Tissue ◽  
Freeze Substitution ◽  
Crystal Formation ◽  
Ice Crystal ◽  
High Pressure Freezing ◽  
Cell Components ◽  
Cold Metal ◽  
Brome Grass

The aim of ultrastructural investigation is to analyze cell architecture and relate a functional role(s) to cell components. It is known that aqueous chemical fixation requires seconds to minutes to penetrate and stabilize cell structure which may result in structural artifacts. The use of ultralow temperatures to fix and prepare specimens, however, leads to a much improved preservation of the cell’s living state. A critical limitation of conventional cryofixation methods (i.e., propane-jet freezing, cold-metal slamming, plunge-freezing) is that only a 10 to 40 μm thick surface layer of cells can be frozen without distorting ice crystal formation. This problem can be allayed by freezing samples under about 2100 bar of hydrostatic pressure which suppresses the formation of ice nuclei and their rate of growth. Thus, 0.6 mm thick samples with a total volume of 1 mm3 can be frozen without ice crystal damage. The purpose of this study is to describe the cellular details and identify potential artifacts in root tissue of barley (Hordeum vulgari L.) and leaf tissue of brome grass (Bromus mollis L.) fixed and prepared by high-pressure freezing (HPF) and freeze substitution (FS) techniques.

High-pressure freezing and freeze substitution of teliospores of the rust fungus Gymnosporangium clavipes

1990 ◽  
Vol 48 (3) ◽  
pp. 692-693
Author(s):  
Robert W. Roberson
Keyword(s):  
High Pressure ◽  
Room Temperature ◽  
Pathogenic Fungus ◽  
Rust Fungus ◽  
Freeze Substitution ◽  
Ice Crystals ◽  
High Pressure Freezing ◽  
Thin Walled Structures ◽  
Cytological Changes ◽  
Dextran Solution

The use of cryo-techniques for the preparation of biological specimens in electron microscopy has led to superior preservation of ultrastructural detail. Although these techniques have obvious advantages, a critical limitation is that only 10-40 μm thick cells and tissue layers can be frozen without the formation of distorting ice crystals. However, thicker samples (600 μm) may be frozen well by rapid freezing under high-pressure (2,100 bar). To date, most work using cryo-techniques on fungi have been confined to examining small, thin-walled structures. High-pressure freezing and freeze substitution are used here to analysis pre-germination stages of specialized, sexual spores (teliospores) of the plant pathogenic fungus Gymnosporangium clavipes C & P.Dormant teliospores were incubated in drops of water at room temperature (25°C) to break dormancy and stimulate germination. Spores were collected at approximately 30 min intervals after hydration so that early cytological changes associated with spore germination could be monitored. Prior to high-pressure freezing, the samples were incubated for 5-10 min in a 20% dextran solution for added cryoprotection during freezing. Forty to 50 spores were placed in specimen cups and holders and immediately frozen at high pressure using the Balzers HPM 010 apparatus.

scholarly journals Neurotoxicity in Marine Invertebrates: An Update

Biology ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 161
Author(s):  
Irene Deidda ◽  
Roberta Russo ◽  
Rosa Bonaventura ◽  
Caterina Costa ◽  
Francesca Zito ◽  
...  
Keyword(s):  
Nervous System ◽  
Marine Invertebrates ◽  
Marine Pollution ◽  
Marine Invertebrate ◽  
High Sensitivity ◽  
Model Systems ◽  
Neural Genes ◽  
Chemical Agents ◽  
Neurotoxic Effects ◽  
Invertebrate Model

Invertebrates represent about 95% of existing species, and most of them belong to aquatic ecosystems. Marine invertebrates are found at intermediate levels of the food chain and, therefore, they play a central role in the biodiversity of ecosystems. Furthermore, these organisms have a short life cycle, easy laboratory manipulation, and high sensitivity to marine pollution and, therefore, they are considered to be optimal bioindicators for assessing detrimental chemical agents that are related to the marine environment and with potential toxicity to human health, including neurotoxicity. In general, albeit simple, the nervous system of marine invertebrates is composed of neuronal and glial cells, and it exhibits biochemical and functional similarities with the vertebrate nervous system, including humans. In recent decades, new genetic and transcriptomic technologies have made the identification of many neural genes and transcription factors homologous to those in humans possible. Neuroinflammation, oxidative stress, and altered levels of neurotransmitters are some of the aspects of neurotoxic effects that can also occur in marine invertebrate organisms. The purpose of this review is to provide an overview of major marine pollutants, such as heavy metals, pesticides, and micro and nano-plastics, with a focus on their neurotoxic effects in marine invertebrate organisms. This review could be a stimulus to bio-research towards the use of invertebrate model systems other than traditional, ethically questionable, time-consuming, and highly expensive mammalian models.

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