scholarly journals Evidence for non-colligative function of small cryoprotectants in a freeze-tolerant insect

2019 ◽  
Vol 286 (1899) ◽  
pp. 20190050 ◽  
Author(s):  
Jantina Toxopeus ◽  
Vladimír Koštál ◽  
Brent J. Sinclair
Keyword(s):  
Fat Body ◽  
Ex Vivo ◽  
Freeze Tolerance ◽  
Field Cricket ◽  
Ice Formation ◽  
Low Molecular Weight ◽  
Manipulative Experiments ◽  
Gryllus Veletis ◽  
Freeze Tolerant

Freeze tolerance, the ability to survive internal ice formation, facilitates survival of some insects in cold habitats. Low-molecular-weight cryoprotectants such as sugars, polyols and amino acids are hypothesized to facilitate freeze tolerance, but their in vivo function is poorly understood. Here, we use a combination of metabolomics and manipulative experiments in vivo and ex vivo to examine the function of multiple cryoprotectants in the spring field cricket Gryllus veletis . Cold-acclimated G. veletis are freeze-tolerant and accumulate myo -inositol, proline and trehalose in their haemolymph and fat body. Injecting freeze-tolerant crickets with proline and trehalose increases survival of freezing to lower temperatures or for longer times. Similarly, exogenous myo -inositol and trehalose increase ex vivo freezing survival of fat body cells from freeze-tolerant crickets. No cryoprotectant (alone or in combination) is sufficient to confer freeze tolerance on non-acclimated, freeze-intolerant G. veletis . Given that each cryoprotectant differentially impacts survival in the frozen state, we conclude that small cryoprotectants are not interchangeable and likely function non-colligatively in insect freeze tolerance. Our study is the first to experimentally demonstrate the importance of non-colligative cryoprotectant function for insect freeze tolerance both in vivo and ex vivo , with implications for choosing new molecules for cryopreservation.

Modeling seasonal changes in intracellular freeze-tolerance of fat body cells of the gall fly Eurosta solidaginis (Diptera, Tephritidae)

1997 ◽  
Vol 200 (1) ◽  
pp. 185-192 ◽  
Author(s):  
V Bennett ◽  
R Lee
Keyword(s):  
Cell Survival ◽  
Seasonal Changes ◽  
Cold Hardiness ◽  
Fat Body ◽  
Freeze Tolerance ◽  
Cellular Level ◽  
Eurosta Solidaginis ◽  
Freeze Tolerant

Although seasonal changes in the freeze-tolerance of third-instar larvae of Eurosta solidaginis have been well documented for the whole organism, the nature of this cold-hardiness at the cellular level has not been examined. Seasonal changes in the survival of fat body cells from E. solidaginis larvae were assessed using fluorescent vital dyes after freezing at -10, -25 or -80 °C for 24 h both in vivo and in vitro. Cells frozen in vitro were frozen with glycerol, with sorbitol (both of which enhanced cell survival) or without cryoprotectants. Both cellular and organismal survival were low in August when larvae were not freeze-tolerant, then increased dramatically during September and October before leveling off from November to January. This observation for cells frozen without cryoprotectants indicates that the cells themselves have adapted. The single most important factor influencing cell survival, as determined by logistic regression modeling, was the time of larval collection, which reflects the level of cold-hardiness achieved by field acclimation. Cells frozen in vivo exhibited greater survival than did those frozen in vitro, even with the addition of cryoprotectants. Since no differences were observed between cells frozen with glycerol or sorbitol, the role of the multi-component cryoprotectant system present in E. solidaginis should be investigated.

scholarly journals Freeze tolerance of Cyphoderris monstrosa (Orthoptera: Prophalangopsidae)

2016 ◽  
Vol 148 (6) ◽  
pp. 668-672 ◽  
Author(s):  
Jantina Toxopeus ◽  
Jacqueline E. Lebenzon ◽  
Alexander H McKinnon ◽  
Brent J. Sinclair
Keyword(s):  
Cold Tolerance ◽  
Freeze Tolerance ◽  
Malpighian Tubules ◽  
Ice Formation ◽  
Freezing And Thawing ◽  
Tolerance Strategy ◽  
Late Instar ◽  
Freeze Tolerant ◽  
Overwintering Survival

AbstractThe great grig, Cyphoderris monstrosa Uhler (Orthoptera: Prophalangopsidae), is a large (20–30 mm, >1 g), nocturnal ensiferan that inhabits montane coniferous forests in northwestern North America. Cyphoderris monstrosa overwinters as a late instar nymphs, but its cold tolerance strategy has not previously been reported. We collected nymphs from near Kamloops, British Columbia, Canada in late spring to determine their cold tolerance strategy. Cyphoderris monstrosa nymphs were active at low temperatures until they froze at −4.6±0.3 °C. The nymphs survived internal ice formation (i.e., are freeze tolerant), had a lethal temperature between −9 °C and −12 °C, and could survive for between five and 10 days at −6 °C. Isolated C. monstrosa gut, Malpighian tubules, and metafemur muscle tissues froze at temperatures similar to whole nymphs, and likely inoculate freezing in vivo. Hemolymph osmolality was 358±51 mOsm, with trehalose and proline comprising ~10% of that total. Glycerol was not detectable in hemolymph from field-fresh nymphs, but accumulated after freezing and thawing. The control of ice formation and presence of hemolymph cryoprotectants may contribute to C. monstrosa freeze tolerance and overwintering survival.

scholarly journals Drivers of plasticity in freeze tolerance in the intertidal mussel Mytilus trossulus

2020 ◽  
Vol 223 (24) ◽  
pp. jeb233478
Author(s):  
Jessica R. Kennedy ◽  
Christopher D. G. Harley ◽  
Katie E. Marshall
Keyword(s):  
Molecular Weight ◽  
Freeze Tolerance ◽  
Cellular Damage ◽  
Low Molecular Weight ◽  
Mytilus Trossulus ◽  
Salinity Acclimation ◽  
Shore Level ◽  
Extreme Stress ◽  
Intertidal Invertebrates ◽  
Freeze Tolerant

ABSTRACTFreezing is an extreme stress to living cells, and so freeze-tolerant animals often accumulate protective molecules (termed cryoprotectants) to prevent the cellular damage caused by freezing. The bay mussel, Mytilus trossulus, is an ecologically important intertidal invertebrate that can survive freezing. Although much is known about the biochemical correlates of freeze tolerance in insects and vertebrates, the cryoprotectants that are used by intertidal invertebrates are not well characterized. Previous work has proposed two possible groups of low-molecular weight cryoprotectants in intertidal invertebrates: osmolytes and anaerobic byproducts. In our study, we examined which group of candidate cryoprotectants correlate with plasticity in freeze tolerance in mussels using 1H NMR metabolomics. We found that the freeze tolerance of M. trossulus varies on a seasonal basis, along an intertidal shore-level gradient, and with changing salinity. Acclimation to increased salinity (30 ppt compared with 15 ppt) increased freeze tolerance, and mussels were significantly more freeze tolerant during the winter. Mussel freeze tolerance also increased with increasing shore level. There was limited evidence that anaerobic byproduct accumulation was associated with increased freeze tolerance. However, osmolyte accumulation was correlated with increased freeze tolerance after high salinity acclimation and in the winter. The concentration of most low molecular weight metabolites did not vary with shore level, indicating that another mechanism is likely responsible for this pattern of variation in freeze tolerance. By identifying osmolytes as a group of molecules that assist in freezing tolerance, we have expanded the known biochemical repertoire of the mechanisms of freeze tolerance.

scholarly journals Laboratory acclimation to autumn-like conditions induces freeze tolerance in the spring field cricket Gryllus veletis (Orthoptera: Gryllidae)

2019 ◽  
Vol 113 ◽  
pp. 9-16 ◽  
Author(s):  
Jantina Toxopeus ◽  
Alexander H McKinnon ◽  
Tomáš Štětina ◽  
Kurtis F. Turnbull ◽  
Brent J. Sinclair
Keyword(s):  
Freeze Tolerance ◽  
Field Cricket ◽  
Gryllus Veletis ◽  
Laboratory Acclimation

scholarly journals Metabolic cost of freeze-thaw and source of CO2 production in the freeze-tolerant cricket Gryllus veletis

2020 ◽  
pp. jeb.234419
Author(s):  
Adam Smith ◽  
Kurtis F. Turnbull ◽  
Julian H. Moulton ◽  
Brent J. Sinclair
Keyword(s):  
Metabolic Rate ◽  
Metabolic Cost ◽  
Field Cricket ◽  
Co2 Production ◽  
System Response ◽  
Freezing And Thawing ◽  
Freeze Thaw ◽  
Metabolic Costs ◽  
Gryllus Veletis ◽  
Freeze Tolerant

Freeze-tolerant insects can survive the conversion of a substantial portion of their body water to ice. While the process of freezing induces active responses from some organisms, these responses appear absent from freeze-tolerant insects. Recovery from freezing likely requires energy expenditure to repair tissues and re-establish homeostasis, which should be evident as elevations in metabolic rate after thaw. We measured carbon dioxide (CO2) production in the spring field cricket (Gryllus veletis) as a proxy for metabolic rate during cooling, freezing and thawing and compared the metabolic costs associated with recovery from freezing and chilling. We hypothesized that freezing does not induce active responses, but that recovery from freeze-thaw is metabolically costly. We observed a burst of CO2 release at the onset of freezing in all crickets that froze, including those killed by either cyanide or an insecticide (thiacloprid), implying that the source of this CO2 was neither aerobic metabolism or a coordinated nervous system response. These results suggest that freezing does not induce active responses from G. veletis, but may liberate buffered CO2 from hemolymph. There was a transient ‘overshoot’ in CO2 release during the first hour of recovery, and elevated metabolic rates at 24, 48 and 72 hours, in crickets that had been frozen compared to crickets that had been chilled (but not frozen). Thus, recovery from freeze-thaw and the repair of freeze-induced damage appears metabolically costly in G. veletis, and this cost persists for several days after thawing.

scholarly journals NOVEL NANOPARTICLES FOR THE ORAL DELIVERY OF LOW MOLECULAR WEIGHT HEPARIN: IN VITRO AND IN VIVO ASSESSMENT

2017 ◽  
Vol 10 (2) ◽  
pp. 254 ◽  
Author(s):  
Nallaguntla Lavanya ◽  
Indira Muzib ◽  
Aukunuru Jithan ◽  
Balekari Umamahesh
Keyword(s):  
Molecular Weight ◽  
Low Molecular Weight Heparin ◽  
Ex Vivo ◽  
In Vitro Release ◽  
Low Molecular Weight ◽  
Scanning Calorimetry ◽  
Ex Vivo Permeation ◽  
Permeation Studies

Objective: The objective of the present study was to prepare and evaluate a novel oral formulation of nanoparticles for the systemic delivery of low molecular weight heparin (LMWH). Methods: Nanoparticles were prepared by polyelectrolyte complexation (PEC) method using polymers sodium alginate and chitosan. Entrapment efficiency of LMWH in nanoparticles was found to be  ̴88%. Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), X‑ray diffraction (XRD), Scanning electron microscopy (SEM)  studies carried for nanoparticles. In vitro release studies were performed for the formulations. Ex vivo permeation studies were performed optimized formulation by using small intestine of rat and in vivo studies were conducted on rat model.Results: In vitro release studies demonstrated that the release of LMWH was negligible in the stomach and high in the small intestine. FTIR has indicated that there is no interaction between the ingredients in nanoparticle. DSC and XRD studies confirmed that the amino groups of chitosan interacted with the carboxylic groups of alginate. Invitro % drug release of 95% was shown by formulation AC5. Ex vivo permeation studies have elucidated that ̴ 73% of LMWH was transported across the epithelium. Nanoparticles have shown enhanced oral bioavailability of LMWH as revealed by 4.5 fold increase in AUC of plasma drug concentration time curve.Conclusion: The results suggest that the nanoparticles prepared can result in targeted delivery of LMWH into systemic circulation via intestinal and colon routes. Novel nanoparticles thus prepared in this study can be considered as a promising delivery system.Keywords: Antifactor Xa activity, Chitosan, Differential scanning calorimetry, Sodium alginate, Low-molecular-weight heparin, Oral bioavailability.

The relationship between the hemorrhagic and antithrombotic properties of low molecular weight heparin in rabbits

Blood ◽  
1982 ◽  
Vol 59 (6) ◽  
pp. 1239-1245 ◽  
Author(s):  
CJ Carter ◽  
JG Kelton ◽  
J Hirsh ◽  
A Cerskus ◽  
AV Santos ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Blood Loss ◽  
Ex Vivo ◽  
Test System ◽  
Venous Stasis ◽  
Low Molecular Weight ◽  
Antithrombotic Effects ◽  
Induced Aggregation

We have compared the hemorrhagic and antithrombotic effects of a low molecular weight (LMW) heparin fraction and standard heparin in rabbits. Similar LMW heparin fractions have antithrombotic effects when tested in animals, but their hemorrhagic effects relative to standard heparin have not been established. Standard porcine mucosal heparin (mol wt 15,000 daltons) was depolymerized by nitrous acid to a low molecular weight fraction (mol wt 4600 daltons). Using equal USP units, the standard and Dep LMW heparin were compared in vitro, ex vivo, and in vivo. In vitro, when diluted in rabbit plasma, the Dep LMW heparin at equivalent anti-Xa activity showed less prolongation of thrombin clotting times or activated partial thromboplastin times. Ex vivo, platelets from rabbits treated with the Dep LMW heparin showed less inhibition of collagen-induced aggregation. The relative hemorrhagic properties of the two heparins were compared in vivo in rabbits using a sensitive blood loss assay, and the antithrombotic properties were compared in a thrombin-induced venous stasis model. By using an optimal threshold heparin dose in each test system, it was possible to demonstrate that equal USP units of Dep LMW heparin caused less blood loss but showed greater antithrombotic activity than standard heparin.

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