Biomedical subjects

D A Wharton

Publications and source records attributed to D A Wharton.

At least 19 recordsLinked to original sources

PubMed2005

Ice-active proteins from the Antarctic nematode Panagrolaimus davidi.

The Antarctic nematode Panagrolaimus davidi has an ice-active protein that shows recrystallization inhibition but no thermal hysteresis. It belongs to a class of ice-active proteins found in a variety of freezing-tolerant organisms that display insignificant levels of thermal hysteresis in the context of the environmental temperatures to which they are exposed. The recrystallization inhibition activity of the P. davidi ice-active protein is present at low concentrations, is relatively heat stable, is affected more by acid than by alkaline pH, is not calcium dependant and is not affected by reagents that target carbohydrate residues or sulphydryl linkages. A hexagonal ice crystal growth form also indicates the presence of an ice-active protein. This protein could have important functions in the survival of intracellular freezing by this organism by controlling the stability of ice after its formation.

Acclimatization↗

PubMed2004

Freezing and cryoprotective dehydration in an Antarctic nematode (Panagrolaimus davidi) visualised using a freeze substitution technique.

The pattern of ice formation during the freezing of Panagrolaimus davidi, an Antarctic nematode that can survive intracellular ice formation, was visualised using a freeze substitution technique and transmission electron microscopy. Nematodes plunged directly into liquid nitrogen had small ice crystals throughout their tissues, including nuclei and organelles, but did not survive. Those frozen at high subzero temperatures showed three patterns of ice formation: no ice, extracellular ice, and intracellular ice. Nematodes subjected to a slow-freezing regime (at -1 degrees C) had mainly extracellular ice (70.4%), with the bulk of the ice in the pseudocoel. Some (24.8%) had no ice within their bodies, due to cryoprotective dehydration. Nematodes subjected to a fast-freezing regime (at -4 degrees C) had intracellular (54%) and extracellular (42%) ice. Intracellular ice was confined to the cytoplasm of cells, with organelles in the spaces in between ice crystals. The survival of nematodes subjected to the fast-freezing regime (53%) was less than those subjected to the slow-freezing regime (92%).

Acclimatization↗

PubMed2003

The environmental physiology of Antarctic terrestrial nematodes: a review.

The environmental physiology of terrestrial Antarctic nematodes is reviewed with an emphasis on their cold-tolerance strategies. These nematodes are living in one of the most extreme environments on Earth and face a variety of stresses, including low temperatures and desiccation. Their diversity is low and declines with latitude. They show resistance adaptation, surviving freezing and desiccation in a dormant state but reproducing when conditions are favourable. At high freezing rates in the surrounding medium the Antarctic nematode Panagrolaimus davidi freezes by inoculative freezing but can survive intracellular freezing. At slow freezing rates this nematode does not freeze but undergoes cryoprotective dehydration. Cold tolerance may be aided by rapid freezing, the production of trehalose and by an ice-active protein that inhibits recrystallisation. P. davidi relies on slow rates of water loss from its habitat, and can survive in a state of anhydrobiosis, perhaps aided by the ability to synthesise trehalose. Teratocephalus tilbrooki and Ditylenchus parcevivens are fast-dehydration strategists. Little is known of the osmoregulatory mechanisms of Antarctic nematodes. Freezing rates are likely to vary with water content in Antarctic soils. Saturated soils may produce slow freezing rates and favour cryoprotective dehydration. As the soil dries freezing rates may become faster, favouring freezing tolerance. When the soil dries completely the nematodes survive anhydrobiotically. Terrestrial Antarctic nematodes thus have a variety of strategies that ensure their survival in a harsh and variable environment. We need to more fully understand the conditions to which they are exposed in Antarctic soils and to apply more natural rates of freezing and desiccation to our studies.

Acclimatization↗

PubMed2003

Dictyocaulus species: cross infection between cattle and red deer.

AIM: To discover whether cross infection between red deer (Cervus elaphus) and cattle is possible with either a bovine isolate of the cattle lungworm, Dictyocaulus viviparus, or with a cervine isolate of the lungworm, Dictyocaulus eckerti which is thought to be maintained primarily in deer. METHOD: Twelve cattle and 12 red deer were reared parasite-free from birth. At 3-4 months of age, half of each species (n=6) were experimentally infected with D. viviparus and the other half with D. eckerti. The course of infection was monitored for 34 days, after which the animals were slaughtered and the lungs removed to assess levels of infection. RESULTS: Faecal larval counts demonstrated that patent Dictyocaulus infections occurred in all groups. At necropsy, adult worms were found in the lungs in all groups except the cattle that were infected with D. eckerti. The largest numbers of adult worms were found in the red deer infected with D. eckerti. CONCLUSION: It was demonstrated that both cattle and red deer could be infected with either D. viviparus or D. eckerti. However, D. eckerti larvae that originated from deer established more successfully in deer and D. viviparus larvae that originated from cattle established more successfully in cattle.

Journal Article↗

PubMed2002

The response of Anisakis larvae to freezing.

Anisakis third stage larvae utilize a variety of fish as intermediate hosts. Uncooked fish are rendered safe for human consumption by freezing. Larvae freeze by inoculative freezing from the surrounding medium but can survive freezing at temperatures down to -10 degrees C. This ability may be aided by the production of trehalose, which can act as a cryoprotectant, but does not involve recrystallization inhibition. Monitoring of fish freezing in commercial blast freezers and under conditions which simulate those of a domestic freezer, indicate that it can take a long time for all parts of the fish to reach a temperature that will kill the larvae. This, and the moderate freezing tolerance of larvae, emphasizes the need for fish to be frozen at a low enough temperature and for a sufficient time to ensure that fish are safe for consumption.

Animals↗

PubMed2001

Water relations during desiccation of cysts of the potato-cyst nematode Globodera rostochiensis.

The loss during desiccation of osmotically active water (OAW), which freezes during cooling to -45 degrees C, and osmotically inactive water (OIW), which remains unfrozen, from the cysts of the potato cyst nematode, Globodera rostochiensis, was determined using differential scanning calorimetry. Exotherms and endotherms associated with non-egg compartments were not detected after 5 min desiccation at 50% relative humidity and 20 degrees C. The pattern of water loss from the cysts indicates that water is lost from compartments outside the eggs first, that nearly all the non-egg water is OAW and that the OIW content of the cyst is contained within the eggs. Water is lost from the eggs only after the OAW content outside the eggs falls below that within the eggs. Both OAW and OIW are lost from the eggs during desiccation but the eggs retain a small amount of OIW. Other animals which survive some desiccation but which are not anhydrobiotic will tolerate the loss of OAW but not the loss of their OIW. Anhydrobiotic animals can survive the loss of both their OAW and a substantial proportion of their OIW.

Adaptation, Physiological↗

PubMed2000

Cold acclimation and cryoprotectants in a freeze-tolerant Antarctic nematode, Panagrolaimus davidi.

Panagrolaimus davidi is a freeze-tolerant Antarctic nematode which survives extensive intracellular freezing. This paper describes the development of culture techniques which provide clean samples, with a high degree of freeze tolerance and in sufficient quantities for the analysis of potential cryoprotectants. Cultures grown at 20 degrees C survived a short-term freezing stress but survival declined with the time spent frozen. Acclimation of cultures at 5 degrees C enhanced the long-term survival of freezing. Starvation, however, reduced the nematode's ability to survive short-term freezing. The principal cryoprotectants detected by gas chromatography were trehalose and glycerol. The levels of trehalose, but not those of glycerol, increased significantly after acclimation. Trehalose may stabilise membranes and protect them against the dehydrating effects of the osmotic stresses resulting from freeze concentration effects but other factors, such as recrystallisation inhibition, may be involved in long-term survival.

Acclimatization↗

PubMed1999

Parasites and low temperatures.

Low temperatures affect the rate of growth, development and metabolism of parasites and when temperatures fall below 0 degrees C may expose the parasite to the potentially lethal risk of freezing. Some parasites have mechanisms, such as diapause, which synchronise their life cycle with favourable seasons and the availability of hosts. Parasites of endothermic hosts are protected from low temperatures by the thermoregulatory abilities of their host. Free-living and off-host stages, however, may be exposed to subzero temperatures and both freezing-tolerant and freeze-avoiding strategies of cold hardiness are found. Parasites of ectothermic hosts may be exposed to subzero temperatures within their hosts. They can rely on the cold tolerance adaptations of their host or they may develop their own mechanisms. Exposure to low temperatures may occur within the carcass of the host and this may be of epidemiological significance if the parasite can be transmitted via the consumption of the carcass.

Adaptation, Physiological↗

PubMed1998

Ultrastructural changes during desiccation of the anhydrobiotic nematode Ditylenchus dipsaci.

Ultrastructural changes during desiccation of the anhydrobiotic nematode Ditylenchus dipsaci were followed and quantified after preparation of material at different levels of hydration using freeze substitution techniques. Some shrinkage was caused by processing in the more hydrated specimens but the changes observed correspond to those observed in live nematodes by light microscopy, indicating that the technique is useful for following changes during desiccation. The overall pattern of changes was a rapid decrease in the magnitude of the measured parameter during the first 5 min of desiccation, followed by a slower rate of decrease upon further desiccation. This was observed in the cuticle, the lateral hypodermal cords and the muscle cells and is consistent with the pattern of water loss of the nematode. The contractile region of the muscle cells, however, proved an exception and the muscle fibres appear to resist shrinkage and packing until water loss becomes severe. The mitochondria swell and then shrink during desiccation, which may indicate disruption of the permeability of the mitochondrial membrane. A decrease in the thickness of the cortical zone was the most prominent change in the cuticle and this may be related to the permeability slump which occurs during the first 5 min of desiccation.

Animals↗

PubMed1997

Differential scanning calorimetry studies on an Antarctic nematode (Panagrolaimus davidi) which survives intracellular freezing.

Differential scanning calorimetry was used to characterize thermal events associated with freezing and melting of suspensions and extracts of Panagrolaimus davidi, an Antarctic nematode which can survive intracellular freezing. Nematode suspensions produced a single freezing exotherm with a shoulder on the peak representing the freezing of the nematodes. A shoulder on the peak of melting endotherms indicates the melting of the nematodes and of the water surrounding them. Exotherms were also detected from individual nematodes mounted in liquid paraffin. The freezing of nematodes was very rapid and in marked contrast to that of freezing-tolerant insects and vertebrates, which take hours or days to freeze. Eighty-two percent of the nematodes' body water froze. High levels of survival were obtained in nematodes exposed to temperatures down to -40 degrees C. No additional thermal events were observed after the freezing event and before the melting of samples cooled to -40 degrees C, indicating no changes in the proportion of body water frozen. Ice nucleating activity is present in nematode suspensions but not in supernatants from nematode extracts. No thermal hysteresis activity was detected in nematode extracts.

Animals↗

PubMed1996

Recrystallization in a freezing tolerant Antarctic nematode, Panagrolaimus davidi, and an alpine weta, Hemideina maori (Orthoptera; Stenopelmatidae).

The ability of haemolymph from the freezing tolerant weta, Hemideina maori, and supernatant from homogenates of the freezing tolerant nematode Panagrolaimus davidi to inhibit the recrystallization of ice was examined using the "splat freezing" technique and annealing on a cryomicroscope stage. There was no recrystallization inhibition in weta haemolymph or in insect ringer controls. Recrystallization inhibition was present in the nematode supernatant but this was destroyed by heating and was absent in controls. P. davidi survives intracellular freezing and recrystallization inhibition may be important for the survival of this stress.

Animals↗

PubMed1996

Osmotic stress effects on the freezing tolerance of the antarctic nematode Panagrolaimus davidi.

The freezing and freezing survival of the Antarctic nematode Panagrolaimus davidi after exposure to solutions of different osmotic concentrations has been examined using a thermoelectric cooling stage and multi-specimen cooling block to see if there is any evidence that freeze-induced desiccation prevents inoculative freezing. The nematodes froze in all the test solutions used (up to 1138 mosmol.l-1) and at all cooling rates and nucleation temperatures tested. Freezing survival was at its maximum in 0.1 mol.l-1 NaCl in artificial tap water after 1 h exposure to the test solution and in artificial tap water after 24 h exposure. Hyperosmotic and hyposmotic stress adversely affected the nematodes' ability to survive freezing. In nonfrozen controls survival declined with increasing osmolality of the test solution. Measurements of the osmolality of water extracted from a variety of moss samples indicate that the nematodes are exposed to an osmotic concentration of about 9 mosmol.l-1 in their natural habitat. This is close to that of artificial tap water. Our experiments, and measurements of freeze concentration effects in the literature, indicate that freeze-induced desiccation is unlikely to prevent inoculative freezing and the survival of nematodes over the winter.

Animals↗

PubMed1995

Desiccation survival of the infective larvae of the insect parasitic nematode, Heterorhabditis zealandica poinar.

Desiccation survival of the insect parasitic nematode Heterorhabditis zealandica upon exposure to various relative humidities on membrane filters and agar plates was determined. Survival was poor once water had been lost from the substrate. A screening experiment indicates that survival would not be improved by adjusting the treatment temperature, the source of the infective larvae, the method of rehydration or the addition of trehalose. Desiccation does not, therefore, appear to be a promising option for the long-term storage of these nematodes.

Animals↗

PubMed1994

A comparison of lungworm faecal larval counts and trichostrongyloid faecal egg counts between red deer (Cervus elaphus) and red deer x wapiti F1 hybrids.

To determine if breed differences in susceptibility to trichostrongyloid and lungworm infection exist, two groups of weaner deer containing seven red deer and red deer x wapiti F1 hybrids were compared using faecal egg counts and faecal larval counts. All animals were run on the same pasture at the same time and treated with the same anthelmintics at the same time. Results indicated that there were significant differences between red deer and red deer x wapiti hybrids, with red deer having higher faecal lungworm counts and red deer x wapiti hybrids having significantly higher faecal egg counts. It is likely that these differences were due to breed. Differences in the efficacy of anthelmintic treatments were also noted between the two groups, with oral oxfendazole being less effective at reducing faecal lungworm counts in red deer X wapiti hybrids than red deer.

Journal Article↗

PubMed1991

Ultrastructural changes associated with exsheathment of infective juveniles of Haemonchus contortus.

Ultrastructural changes associated with exsheathment of the infective juveniles of Haemonchus contortus are described. Hyaline ring formation occurs associated with annulations 10 and 20 and consists of the dissolution of the basal zone and the inner electron-dense layer resulting in the rupture of the sheath. These changes are consistent with the action of a protease-exsheathing enzyme. There is a significant reduction associated with exsheathment in the size of the excretory cells and the number of excretory granules contained within them. No changes in the oesophagus were observed associated with exsheathment. These, and the observations of earlier workers, suggest that it is the excretory cells and not the oesophagus which are the source of exsheathing fluid during exsheathment.

Analysis of Variance↗

PubMed1991

Freeze-substitution techniques for preparing nematodes for scanning electron microscopy.

The effect of different substitution times, temperatures and the incorporation of fixatives on the preservation of three species of nematode for scanning electron microscopy by freeze substitution with methanol, followed by critical point drying, is investigated. Hammerschmidtiella diesingi adults and Trichostrongylus colubriformis infective juveniles were successfully preserved using methanol at 253 K as the substitution medium. Preservation deteriorated with long substitution times, suggesting the extraction of material and that substitution times should be kept as brief as possible. Panagrolaimus davidi was not successfully preserved using pure methanol, but preservation was improved by using fixatives in the substitution medium, the best results being obtained with 1% OsO4/3% glutaraldehyde in methanol. A substitution temperature of 193 K did not give any improvement in preservation. The differences in the quality of preservation between the three species may be due to the relative ability of the cuticle to withstand collapse during critical point drying. Chemical fixation using cold fixative resulted in the retention of a natural posture but poor preservation, whereas hot fixatives resulted in good preservation but the loss of a natural posture. Freeze substitution in methanol may prove useful in the preparation of specimens possessing cuticles or cell walls which have sufficient strength to withstand the drying process (e.g. arthropods, plants, fungi, nematodes). More delicate specimens may require the incorporation of fixatives into the substitution medium or conventional fixation.

Animals↗

PubMed1990

Anhydrobiosis in the infective juveniles of Trichostrongylus colubriformis (Nematoda: Trichostrongylidae).

The infective juveniles of Trichostrongylus colubriformis can survive exposure to 0% relative humidity after desiccation at higher relative humidities. During rehydration there is a lag phase before recovery. The infective juveniles are thus capable of anhydrobiosis. Removal of the sheath does not affect desiccation survival but does affect the duration of the lag phase. Morphological changes during the lag phase are described. The appearance of birefringence in the muscle cells during the anomalous shrinkage which occurs during the lag phase is considered to reflect physiological changes necessary for the recovery of normal muscle function.

Animals↗

PubMed1990

The use of computer-aided learning for teaching parasite life cycles.

The difficulty of presenting parasite life cycles in a textual or diagrammatic form is discussed and the use of computer-aided learning to solve this is described. The design and use of a Hypercard stack for teaching parasite life cycles on an Apple Macintosh microcomputer are outlined.

Animals↗