Freeze or dehydrate: only two options for the survival of subzero temperatures in the arctic enchytraeid<Emphasis Type="Italic"> Fridericia ratzeli</Emphasis>

Hygrophilic soil animals, like enchytraeids, overwintering in frozen soil are unlikely to base their cold tolerance on supercooling of body fluids. It seems more likely that they will either freeze due to inoculative freezing, or dehydrate and adjust their body fluid melting point to ambient temperature as has been shown for earthworm cocoons and Collembola. In the present study we tested this hypothesis by exposing field-collected adult Fridericia ratzeli from Disko, West Greenland, to freezing temperatures under various moisture regimes. When cooled at –1 °C min^–1 under dry conditions F. ratzeli had a mean temperature of crystallisation (T_c) of –5.8 °C. However, when exposed to temperatures above standard T_c for 22 h, at –4 °C, most individuals (90%, n= 30) remained unfrozen. Slow cooling from –1 °C to –6 °C in vials where the air was in equilibrium with the vapour pressure of ice resulted in freezing in about 65% of the individuals. These individuals maintained a normal body water content of 2.7–3.0 mg mg^–1 dry weight and had body fluid melting points of about –0.5 °C with little or no change due to freezing. About 35% of the individuals dehydrated drastically to below 1.1 mg mg^–1 dry weight at –6 °C, and consequently had lowered their body fluid melting point to ca. –6 °C at this time. Survival was high in both frozen and dehydrated animals at –6 °C, about 60%. Approximately 25% of the animals (both frozen and dehydrated individuals) had elevated glucose concentrations, but the mean glucose concentration was not increased to any great extent in any group due to cold exposure. The desiccating potential of ice was simulated using aqueous NaCl solutions at 0 °C. Water loss and survival in this experiment were in good agreement with results from freezing experiments. The influence of soil moisture on survival and tendency to dehydrate was also evaluated. However, soil moisture ranging between 0.74 g g^–1 and 1.15 g g^–1 dry soil did not result in any significant differences in survival or frequency of dehydrated animals even though the apparent wetness and structure of the soil was clearly different in these moisture contents.Abbreviations DW dry weight - FW fresh weight - MP melting point - RH relative humidity - Tc crystallisation temperatures - WC water contentCommunicated by I.D. Hume     

The distribution,structure, and composition of freshwater ice deposits in Bolivian salt lakes

Freshwater ice deposits are described from seven, high elevation (4117–4730 m), shallow (mean depth <30 cm), saline (10–103 g l^-1) lakes in the southwestern corner of Bolivia. The ice deposits range to several hundred meters in length and to 7 m in height above the lake or playa surface. They are located near the lake or salar margins; some are completely surrounded by water, others by playa deposits or salt crusts. Upper surfaces and sides of the ice deposits usually are covered by 20–40 cm of white to light brown, dry sedimentary materials. Calcite is the dominant crystalline mineral in these, and amorphous materials such as diatom frustules and volcanic glass are also often abundant.Beneath the dry overburden the ice occurs primarily as horizontal lenses 1–1000 mm thick, irregularly alternating with strata of frozen sedimentary materials. Ice represents from 10 to 87% of the volume of the deposits and yields freshwater (TFR <3 g l^-1) when melted. Oxygen isotope ratios for ice are similar to those for regional precipitation and shoreline seeps but much lower than those for the lakewaters. Geothermal flux is high in the region as evidenced by numerous hot springs and deep (3.0–3.5 m) sediment temperatures of 5–10°C. This flux is one cause of the present gradual wasting away of these deposits. Mean annual air temperatures for the different lakes probably are all in the range of -2 to 4°C, and mean midwinter temperatures about 5°C lower. These deposits apparently formed during colder climatic conditions by the freezing of low salinity porewaters and the building up of segregation ice lenses.     

Temperature and water content effects on the viscoelastic behavior of<Emphasis Type="Italic"> Tilia americana</Emphasis> (Tiliaceae) sapwood

The effects of temperature and water content on the viscoelasticity of living and dehydrated Tilia americana sapwood were examined using transient creep (time- and load-dependent deformation) tests under sustained bending loads. Creep tests were performed at 21.1°C and –20.5°C to determine the magnitudes and types of strains in living and dehydrated samples. Temperature had no effect on the creep rate of living sapwood. However, the creep rate of dehydrated samples at –20.5°C was significantly faster than that at 21.1°C. Regardless of temperature, sapwood had a faster creep rate than dehydrated samples. With small bending loads, the residual strains in sapwood were larger at 21.1°C compared to –20.5°C. Temperature did not significantly affect the residual strains in dehydrated samples. For small bending loads, frozen sapwood recovered all residual creep strains when thawed. With larger loads, residual and plastic (permanent) strains increased. We speculate that ice formation in cell lumens partially dehydrates (and thus stiffens and strengthens) cell wall materials and prevents cell wall buckling and elastic restoration after unloading. However, when thawed, sapwood can elastically restore its original configuration, provided it is not excessively bent (by ice or snow accumulations) when frozen.     

UV radiation and potential biological effects beneath the perennial ice cover of an antarctic lake

High-resolution spectral scans of solar ultraviolet radiation (UVR) were obtained directly beneath the 4.0–5.0 m thick, perennial ice cover of Lake Hoare, South Victoria Land, Antarctica. Both UVA (320–400 nm) and UVB (280–320 nm) radiation were detectable beneath the ice using a diver-deployed, underwater scanning spectroradiometer which permitted accurate measurement in the 280–340 nm range, while avoiding effects of surface shading and/or hole effects. UVR at wavelengths <310 nm was not detectable below the ice. This lower wavelength UVB appears to penetrate the Lake Hoare ice to depths of no more than 1.5 m during relatively cloud-free austral summer days. Based upon estimated biologically effective UVR dosages and DNA dosimeter data, exposure of benthic and planktonic microbes to the UVR encountered immediately beneath the ice is unlikely to inhibit microbial metabolism. Although waters of oligotrophic antarctic lakes are highly transparent to UVR, the thick, high scattering and optically dense ice covers on many of these lakes offers organisms a degree of protection largely unavailable in temperate and tropical systems. Thinning or complete loss of these overlying ice covers is likely to have major consequences for the structure of antarctic lake microbial communities.     

Effect of accelerated electron beam on mechanical properties of human cortical bone: influence of different processing methods

Accelerated electron beam (EB) irradiation has been a sufficient method used for sterilisation of human tissue grafts for many years in a number of tissue banks. Accelerated EB, in contrast to more often used gamma photons, is a form of ionizing radiation that is characterized by lower penetration, however it is more effective in producing ionisation and to reach the same level of sterility, the exposition time of irradiated product is shorter. There are several factors, including dose and temperature of irradiation, processing conditions, as well as source of irradiation that may influence mechanical properties of a bone graft. The purpose of this study was to evaluate the effect e-beam irradiation with doses of 25 or 35?kGy, performed on dry ice or at ambient temperature, on mechanical properties of non-defatted or defatted compact bone grafts. Left and right femurs from six male cadaveric donors, aged from 46 to 54?years, were transversely cut into slices of 10?mm height, parallel to the longitudinal axis of the bone. Compact bone rings were assigned to the eight experimental groups according to the different processing method (defatted or non-defatted), as well as e-beam irradiation dose (25 or 35?kGy) and temperature conditions of irradiation (ambient temperature or dry ice). Axial compression testing was performed with a material testing machine. Results obtained for elastic and plastic regions of stress-strain curves examined by univariate analysis are described. Based on multivariate analysis, including all groups, it was found that temperature of e-beam irradiation and defatting had no consistent significant effect on evaluated mechanical parameters of compact bone rings. In contrast, irradiation with both doses significantly decreased the ultimate strain and its derivative toughness, while not affecting the ultimate stress (bone strength). As no deterioration of mechanical properties was observed in the elastic region, the reduction of the energy absorption capacity of irradiated bone rings apparently resulted from changes generated by irradiation within the plastic strain region.     

A Novel Method of Estimating Dose Responses for Polymer Gels Using Texture Analysis of Scanning Electron Microscopy Images

Polymer gels are regarded as a potential dosimeter for independent validation of absorbed doses in clinical radiotherapy. Several imaging modalities have been used to convert radiation-induced polymerization to absorbed doses from a macro-scale viewpoint. This study developed a novel dose conversion mechanism by texture analysis of scanning electron microscopy (SEM) images. The modified N-isopropyl-acrylamide (NIPAM) gels were prepared under normoxic conditions, and were administered radiation doses from 5 to 20 Gy. After freeze drying, the gel samples were sliced for SEM scanning with 50×, 500×, and 3500× magnifications. Four texture indices were calculated based on the gray level co-occurrence matrix (GLCM). The results showed that entropy and homogeneity were more suitable than contrast and energy as dose indices for higher linearity and sensitivity of the dose response curves. After parameter optimization, an R ² value of 0.993 can be achieved for homogeneity using 500× magnified SEM images with 27 pixel offsets and no outlier exclusion. For dose verification, the percentage errors between the prescribed dose and the measured dose for 5, 10, 15, and 20 Gy were −7.60%, 5.80%, 2.53%, and −0.95%, respectively. We conclude that texture analysis can be applied to the SEM images of gel dosimeters to accurately convert micro-scale structural features to absorbed doses. The proposed method may extend the feasibility of applying gel dosimeters in the fields of diagnostic radiology and radiation protection.     

Validation of 15 kGy as a radiation sterilisation dose for bone allografts manufactured at the Queensland Bone Bank: application of the VD<Subscript>max</Subscript> 15 method

Background ISO 11137-2006 (ISO 11137-2a 2006) provides a VD_max 15 method for substantiation of 15 kGy as radiation sterilisation dose (RSD) for health care products with a relatively low sample requirement. Moreover, the method is also valid for products in which the bioburden level is less than or equal to 1.5. In the literature, the bioburden level of processed bone allografts is extremely low. Similarly, the Queensland Bone Bank (QBB) usually recovers no viable organisms from processed bone allografts. Because bone allografts are treated as a type of health care product, the aim of this research was to substantiate 15 kGy as a RSD for frozen bone allografts at the QBB using method VD_max 15—ISO 11137-2: 2006 (ISO 11137-2e, Procedure for method VD_max 15 for multiple production batches. Sterilisation of health care products – radiation – part 2: establishing the sterilisation dose, 2006; ISO 11137-2f, Procedure for method VD_max 15 for a single production batch. Sterilisation of health care products – radiation – part 2: establishing the sterilisation dose, 2006). Materials 30 femoral heads, 40 milled bone allografts and 40 structural bone allografts manufactured according to QBB standard operating procedures were used. Method Estimated bioburdens for each bone allograft group were used to calculate the verification doses. Next, 10 samples per group were irradiated at the verification dose, sterility was tested and the number of positive tests of sterility recorded. If the number of positive samples was no more than 1, from the 10 tests carried out in each group, the verification was accepted and 15 kGy was substantiated as RSD for those bone allografts. Results The bioburdens in all three groups were 0, and therefore the verification doses were 0 kGy. Sterility tests of femoral heads and milled bones were all negative (no contamination), and there was one positive test of sterility in the structural bone allograft. Accordingly, the verification was accepted. Conclusion Using the ISO validated protocol, VD_max 15, 15 kGy was substantiated as RSD for frozen bone allografts manufactured at the QBB.     

Normoxic polymer gel dosimetry using less toxic monomer of N-isopropyl acrylamide and X-ray computed tomography for radiation therapy applications

Background

Polymer gel dosimetry has been used extensively in radiation therapy for its capability in depicting a three dimensional view of absorbed dose distribution. However, more studies are required to find less toxic and more efficient polymers for application in radiotherapy dosimetry.

Aim

The purpose of this work was to evaluate the N-isopropyl acrylamide (NIPAM) gel dosimetric characteristics and optimize the protocol for X-ray computed tomography (CT) imaging of gel dosimeters for radiation therapy application.

Material and methods

A polymer gel dosimeter based on NIPAM monomer was prepared and irradiated with ⁶⁰Co photons. The CT number changes following irradiation were extracted from CT images obtained with different sets of imaging parameters.

Results

The results showed the dose sensitivity of ΔN_CT (H) = 0.282 ± 0.018 (H Gy⁻¹) for NIPAM gel dosimeter. The optimized set of imaging exposure parameters was 120 kV_p and 200 mA with the 10 mm slice thickness. Results of the depth dose measurement with gel dosimeter showed a great discrepancy with the actual depth dose data.

Conclusion

According to the current study, NIPAM-based gel dosimetry with X-ray CT imaging needs more technical development and formulation refinement to be used for radiation therapy application.     

Formation of extracellular and intracellular ice during warming of vitrified mouse morulae and its effect on embryo survival

In vitrified solutions, ice can form during warming if the concentration of the cryoprotectant is insufficient. For the cryopreservation of cells, ice is innocuous when it remains outside the cell, but intracellular ice (ICI) is lethal. We tried to estimate the conditions in which ICI forms in vitrified mouse morulae during warming. The solutions for the experiments (EFS10–EFS50) contained 10–50% ethylene glycol plus Ficoll plus sucrose. When vitrified EFS20, EFS30, and EFS40 were kept at −80 °C, they remained transparent after 3 min, but turned opaque after 60 min (EFS20, EFS30) or 24 h (EFS40). Morulae were vitrified with EFS solutions after exposure for 30–120 s at 25 °C. They were warmed by various methods and survival was assessed in culture. After rapid warming (control), survival was high with EFS30 (79–93%) and EFS40 (96–99%). After slow warming, survival decreased with both EFS30 (48–62%) and EFS40 (44–64%). This must be from the formation of ICI. To examine the temperature at which ICI formed during slow warming, vitrified embryos were kept at various sub-zero temperatures during warming. Survival with EFS30 and EFS40 decreased on keeping samples for 3 min at −80 (25–75%), −60 (7–49%), −40 (0–41%), or −20 °C (26–60%). When samples were kept at −80 °C for 24 h, the survival decreased to 0–14%. These results suggest that ICI forms at a wide range of temperatures including −80 and −20 °C, more likely between −60 and −40 °C, and the ice forms not only quickly but also slowly.     

Effect of gamma irradiation on mechanical properties of human cortical bone: influence of different processing methods

The secondary sterilisation by irradiation reduces the risk of infectious disease transmission with tissue allografts. Achieving sterility of bone tissue grafts compromises its biomechanical properties. There are several factors, including dose and temperature of irradiation, as well as processing conditions, that may influence mechanical properties of a bone graft. The purpose of this study was to evaluate the effect of gamma irradiation with doses of 25 or 35?kGy, performed on dry ice or at ambient temperature, on mechanical properties of non-defatted or defatted compact bone grafts. Left and right femurs from six male cadaveric donors aged from 46 to 54?years, were transversely cut into slices of 10?mm height, parallel to the longitudinal axis of the bone. Compact bone rings were assigned to the eight experimental groups according to the different processing method (defatted or non-defatted), as well as gamma irradiation dose (25 or 35?kGy) and temperature conditions of irradiation (ambient temperature or dry ice). Axial compression testing was performed with a material testing machine. Results obtained for elastic and plastic regions of stress-strain curves examined by univariate analysis are described. Based on multivariate analysis it was found that defatting of bone rings had no significant effect on any mechanical parameter studied, whereas irradiation with both doses decreased significantly the ultimate strain and its derivative toughness. The elastic limit and resilience were significantly increased by irradiation with the dose 25?kGy, but not 35?kGy, when the time of irradiation was longer. Additionally, irradiation at ambient temperature decreased maximum load, elastic limit, resilience, and ultimate stress. As strain in the elastic region was not affected, decreased elastic limit resulted in lower resilience. The opposite phenomenon was observed in the plastic region, where in spite of the lower ultimate stress, the toughness was increased due to the increase in the ultimate strain. The results of our study suggest that there may be an association between mechanical properties of bone tissue grafts and the damage process of collagen structure during gamma irradiation. This collagen damage in cortical bone allografts containing water does not depends on the temperature of irradiation or defatting during processing if dose of gamma irradiation does not exceed 35?kGy.     

Wheat tissues freeze-etched during exposure to extracellular freezing: distribution of ice

Pieces excised from leaf bases and laminae of seedlings of Triticum aestivum L. cv. Lennox were slowly frozen, using a specially designed apparatus, to temperatures between 2° and 14° C. These treatments ranged from non-damaging to damaging, based on ion-leakage tests to be found in the accompanying report (Pearce and Willison 1985, Planta 163, 304–316). The frozen tissue pieces were then freeze-fixed by rapidly cooling them, via melting Freon, to liquid-nitrogen temperature. The tissue was subsequently prepared for electron microscopy by freeze-etching. Ice crystals formed during slow freezing would tend to be much larger than those formed during subsequent freeze-fixation. Ice crystals surrounding the excised tissues were much larger in the frozen than in the control tissues (the latter rapidly freeze-fixed from room temperature). Large ice crystals were present between cells of frozen laminae and absent from controls. Intercellular spaces were infrequent in control leaf bases and no ice-filled intercellular spaces were found in frozen leaf bases. Intracellular ice crystals were smaller in frozen tissues than in controls. It is concluded that all ice formation before freeze-fixation was extracellular. This extracellular ice was either only extra-tissue (leaf bases), or extra-tissue and intercellular (laminae). Periplasmic ice was sometimes present, in control as well as slowly frozen tissues, and the crystals were always small; thus they were probably formed during freeze-fixation rather than during slow freezing. The plasma membrane sometimes showed imprints of cell-wall microfibrils. These were less abundant in leaf bases at 8° C than in controls, and were present on only a minority of plasma membranes from laminae. Therefore, extracellular ice probably did not compress the cells substantially, and changes in cell size and shape were possibly primarily a result of freezing-induced dehydration. Fine-scale distortions (wrinkles) in the plasma membrane, while absent from controls, were present, although only rarely, in both damaged and non-damaged tissues; they were therefore ice-induced but not directly related to the process of damage.     

Vorläufige Ergebnisse von Untersuchungen an eingefrorenen und bei tiefen Temperaturen bestrahlten Lymphozyten

Zusammenfassung Lymphozytenkulturen werden nach Zugabe von Glyzerin als Gefrierschutzsubstanz zunächst gemeinsam mit ungefähr 1 °C/min Abkühlgeschwindigkeit bis zum vollständigen Erstarren eingefroren und dann mit verschiedener Geschwindigkeit ( 200 °C pro min und 3 bis 5 °C/min) auf verschieden tiefe Temperaturen (–22, –80 und –196 °C) gebracht. Alle eingefrorenen Proben weisen nach 24 h Kulturdauer eine kleinere, nach 48 h Kulturdauer eine wesentlich höhere Mitoserate als die Kontrollgruppe (unbehandelte, nicht eingefrorene Plasmaproben) auf. In den schnell auf –196 °C abgekühlten Proben wurden in beiden Fällen (24 h und 48 h Kulturdauer) keine Metaphaseplatten gefunden. Die Zellkonzentrationen waren nur bei den schnell auf –196 °C abgekühlten Kulturen stark verringert. Die Chromosomenaberrationsrate der eingefrorenen Kulturen ist nicht signifikant erhöht.

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Preliminary results of investigations of lymphocytes, frozen and irradiated at low temperaturesI. The effects of cooling rate, low temperature, and incubation time on frozen lymphocytes

Summary Peripheral human lymphocytes were cooled to the temperature of solidification at a rate of less than 1 °C/min using glycerol as a protective agent against the effects of freezing. After solidification at temperatures of –10 to –15 °C and cooling to about –22 °C one group of the samples was thawn and the others were cooled rapidly to –80 or –196 °C by immersion into solid carbon dioxide or liquid nitrogen. Other samples of the frozen cell suspension were cooled down to the same temperatures much slower at a cooling rate of 3 to 5 °C/min. After rapid thawing in a 25 °C water bath the cell suspensions were removed from glycerol and cultured for 24 or 48 h before stopping mitoses by adding colchicine. The samples frozen at –22 °C, the samples cooled both rapidly and slowly to –79 °C, and the ones cooled slowly to –196 °C showed a lower rate of mitosis when colchicine was added after 24 h and a significantly higher rate of mitosis after 48 h of incubation before adding colchicine as compared to the controls (untreated, unfrozen plasma). In the culture frozen rapidly to –196 °C no metaphases could be found. The cell concentrations before and after freezing showed no significant differences except those of the culture frozen rapidly to –196 °C. The chromosome aberration rate is not significantly increased.

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Herrn Prof. Dr. H. A. Künkel zum 60. Geburtstag gewidmet.     

Beneficial effects of enhanced UV-B radiation under field conditions: improvement of needle water relations and survival capacity of Pinus pinea L. seedlings during the dry Mediterranean summer

The possible mechanism(s) by which supplemental UV-B radiation alleviates the adverse effects of summer drought in Mediterranean pines (Petropoulou et al. 1995) were investigated with seedlings of Pinus pinea. Plants received ambient or ambient plus supplemental UV-B radiation (biologically equivalent to a 15% ozone depletion over Patras, 38.3° N, 29.1° E) and natural precipitation or additional irrigation. Treatments started on 1 February, 1994 and lasted up to the end of the dry period (29 September). In well-watered plants, UV-B radiation had no influence on photosystem II photochemical efficiency and biomass accumulation. Water stressed plants suffered from needle loss and reduced photosystem II photochemical efficiency during the summer. These symptoms, however, were less pronounced in plants receiving supplemental UV-B radiation, resulting in higher total biomass at plant harvest. Laboratory tests showed that enhanced UV-B radiation did not improve the tolerance of photosystem II against drought, high light, high temperature and oxidative stress. Enhanced UV-B radiation, however, improved the water economy of water stressed plants, as judged by measurements of needle relative water content. In addition, it caused an almost two-fold increase of cuticle thickness. No such UV-B radiation effects were observed in well-watered pines. The results indicate that the combination of water stress and UV-B radiation may trigger specific responses, enabling the plants to avoid excessive water loss and, thereby, maintain a more efficient photosynthetic apparatus during the summer. The extent of this apparently positive UV-B radiation effect would depend on the amount of summer precipitation. Abbreviations: DW – dry weight, F_v/F_m – ratio of variable to maximum fluorescence, A ₃₀₀ – absorbance at 300 nm, PAR – photosynthetically active radiation, PS II – photosystem II, RWC – relative water content, TCA – trichloroacetic acid, UV-B_BE – biologically effective ultraviolet-B radiation     

Effect of low dose and moderate dose gamma irradiation on the mechanical properties of bone and soft tissue allografts

The increased use of allograft tissue for musculoskeletal repair has brought more focus to the safety of allogenic tissue and the efficacy of various sterilization techniques. Gamma irradiation is an effective method for providing terminal sterilization to biological tissue, but it is also reported to have deleterious effects on tissue mechanics in a dose-dependent manner. At irradiation ranges up to 25 kGy, a clear relationship between mechanical strength and dose has yet to be established. The aim of this study was to investigate the mechanical properties of bone and soft tissue allografts, irradiated on dry ice at a low absorbed dose (18.3–21.8 kGy) and a moderate absorbed dose (24.0–28.5 kGy), using conventional compressive and tensile testing, respectively. Bone grafts consisted of Cloward dowels and iliac crest wedges, while soft tissue grafts consisted of patellar tendons, anterior tibialis tendons, semitendinosus tendons, and fascia lata. There were no statistical differences in mechanical strength or modulus of elasticity for any graft irradiated at a low absorbed dose, compared to control groups. Also, bone allografts and two soft tissue allografts (anterior tibialis and semitendinosus tendon) that were irradiated at a moderate dose demonstrated similar strength and modulus of elasticity values to control groups. The results of this study support the use of low dose and moderate dose gamma irradiation of bone grafts. For soft tissue grafts, the results support the use of low dose irradiation.     

Studies on the rust,Maravalia cryptostegiae,a potential biological control agent of rubber-vine weed,Cryptostegia grandiflora (Asclepiadaceae: Periplocoideae), in Australia,II: Infection

The parameters which govern infection of rubber-vine weed by the rustMaravalia cryptostegiae were investigated. The infection process, from appressorial formation to sporulation, is described and illustrated. Uredinioid teliospores have an optimum temperature range for germination at 22–27 °C, both in vitro and in vivo. However, germination on the rubber-vine leaf was more than double (81–92%) that in the absence of the host, and appressoria were formed only in vivo. An optimum temperature of 20–22°C and a dew period of 12 hours or more gave the highest level of infection as measured by sporulation density. The latent period from inoculation to pustule formation decreased with increasing temperature; the shortest period (8–11 days) being recorded at 25–27°C. At the lower temperatures (18°C), this was significantly extended (19–21 days). Four successive inoculations significantly reduced plant height and dry weight, although a compensatory growth flush occurred after the third inoculation. The addition of cryoprotectants had a negative affect on spore viability and subsequent infectivity. Cooling dry spores to –196°C at the rate of 10°C min^–1 gave the best results, with high germination (93–65%) up to 8 days after thawing.     

Asymmetric somatic cell hybridization in plants

Summary An investigation into the possible application of UV radiation as a pretreatment for the donor cells in asymmetric plant cell hybridization protocols has been carried out. A comparison was made between the effects of UV doses in the range 700–4200 J/m² and those of ⁶⁰Co gamma radiation over the range 0.15–1 kGy on Beta vulgaris suspension cell protoplasts. The investigation had two aspects. Firstly, alterations to cell physiology (cell wall resynthesis, viability, division and colony formation) in irradiated protoplasts were examined during a 4-week culture period. Results have indicated that a dose of 700 J/m² UV is necessary to prevent further cell division and colony formation in these cells. A dose of 0.15 kGy gamma radiation generally prevented colony formation, although some early cell division did occur (as was also observed even after 0.45 kGy had been applied). Membrane integrity, as measured after 6 days, using fluorescein diacetate staining, was not affected by either treatment within the dose ranges applied. Secondly, denaturing (alkaline) gel electrophoresis, in association with a pulsed field gel DNA preparation technique, was used to determine the degree of in vivo DNA damage following the radiation treatments. After UV radiation, considerable fragmentation of the DNA was observed, the extent of which was dose-dependent. Gamma radiation, however, appeared to result in fewer DNA lesions, with only the 1 kGy treatment revealing a pattern significantly altered from that of the control. These results augur well for the potential use of UV radiation in asymmetric fusion experiments.     

Cold tolerance of Littorinidae from southern Africa: intertidal snails are not constrained to freeze tolerance

All intertidal gastropods for which cold tolerance strategies have been assessed have been shown to be freeze tolerant. Thus, freeze tolerance is considered an adaptation to the intertidal environment. We investigated the cold tolerance strategies of three species of subtropical and temperate snails (Gastropoda: Littorinidae) to determine whether this group is phylogenetically constrained to freeze tolerance. We exposed dry acclimated and wet rehydrated snails to low temperatures to determine temperature of crystallisation (Tc), lower lethal temperature and LT₅₀ and to examine the relationship between ice formation and mortality. Tc was lowest in dry Afrolittorina knysnaensis (–13.6±0.4 °C), followed by dry Echinolittorina natalensis (–10.9±0.2 °C) and wet A. knysnaensis (–10.2±0.2 °C). The Tc of both A. knysnaensis and E. natalensis increased with rehydration, whereas Tc of dry and wet Afrolittorina africana did not differ (–9.6±0.2 and –9.0±0.2 °C respectively). Wet snails of all species exhibited no or low survival of inoculative freezing, whereas dry individuals of A. knysnaensis could survive subzero temperatures above –8 °C when freezing was inoculated with ice. In the absence of external ice, Afrolittorina knysnaensis employs a freeze-avoidance strategy of cold tolerance, the first time this has been reported for an intertidal snail, indicating that there is no family-level phylogenetic constraint to freeze tolerance. Echinolittorina natalensis and A. africana both showed pre-freeze mortality and survival of some internal ice formation, but were not cold hardy in any strict sense.     

Effect of incubation temperature on green sturgeon embryos, <Emphasis Type="Italic">Acipenser medirostris</Emphasis>

Regulation of river flow and the amount of winter rainfall are the major factors affecting the water temperature of the spawning grounds, for green sturgeon in the Klamath River. During the primary spawning period of green sturgeon, mid-April to June, the water temperature may vary from 8 to 21°C. To estimate the potential implications of this modified thermal regime, we examined the survival and development in three progeny groups of green sturgeon embryos from zygote to hatch, at constant incubation temperatures (11–26°C). Temperatures 23–26°C affected cleavage and gastrulation and all died before hatch. Temperatures 17.5–22°C were suboptimal as an increasing number of embryos developed abnormally and hatching success decreased at 20.5–22°C, although the tolerance to these temperatures varied between progenies. The lower temperature limit was not evident from this study, although hatching rate decreased at 11°C and hatched embryos were shorter, compared to 14°C. The mean total length of hatched embryos decreased with increasing temperature, although their wet and dry weight remained relatively constant. We concluded that temperatures 17–18°C may be the upper limit of the thermal optima for green sturgeon embryos, and that the river thermal regime during dry years may affect green sturgeon reproduction.     

Temperature conditions enhancing resting egg production of the euryhaline rotifer Brachionus plicatilis O. F. Müller (Kamiura strain)

We examined the effect of low temperature treatment(12°C), followed by transfer to highertemperature (25°C), on resting egg formation ofthe rotifer Brachionus plicatilis, Kamiurastrain. This strain has been mass cultured as livefeed at Kamiura Station (Japan Sea FarmingAssociation) for 9 years at 20°C without theappearance of sexual reproductive stages.After preculture in 20 l of 27 seawater at 12°C for 0, 10, 20, and 30 days,rotifers were inoculated into 0.5 l mass cultures andcultured at 25°C for 7–9 days. The inoculationdensities were changed from 20 to 400 ind. ml^–1,depending on mixis rate. Condensed and frozen Nannochloropsis oculata was fed to rotifers at thefeeding rate of 0.14 µg (dry weight)rotifer^–1day^–1. The control was cultured at12°C for the entire 36 day experiment. No mixisappeared and no resting eggs were produced when thelow temperature treatment was 0 or 10 days. However,mixis rates reached 50-60% after 20 or 30 days ofexposure to 12°C. The number of resting eggsproduced in these treatments reached 25,500 about 13 times higher than the control. Our resultssuggest that low temperature stimulated mictic femaleproduction and the transfer to the high temperatureaccelerated resting egg formation. This method may beuseful for producing resting eggs of rotifer strainsthat lack sexual reproduction in the common culturecondition at larval rearing facilities.     

Ammonia-nitrogen excretion in Daphnia pulex

Ammonia-nitrogen excretion rates were measured in natural summer and cultured populations of Daphnia pulex from Silver Lake, Clay County, Minnesota, USA during 1973. The mean rate of ammonia-nitrogen excretion for the summer populations was 0.20 µg N animal^–1 day^–1 or 5.11 µg N mg^–1 dry body weight day^–1 (N = 80) measured at 15°, 20°, and 25°C. These rates appear to be temperature and weight dependent, but they are probably affected by factors other than temperature and dry body weight. Ammonia-nitrogen excretion rates of Daphnia pulex cultured on Chlamydomonas reinhardi yielded the following relationship with temperature: Log₁₀E = (0.061) T 1.773, where E is µg N animal^–1 day^–1 and T is temperature °C. The ammonia-nitrogen excretion on a mg^–1 dry body weight day^–1 basis was related to temperature according to the following similar expression Log₁₀E = (0.043) T + 0.153, where E is µg N mg^–1 dry body weight day^–1, and T is temperature °C. The length-weight relationship of Daphnia pulex for the summer populations (N = 1583) was log₁₀W = (0.526) Log₁₀L + 1.357, where W is weight in µg and L is length in mm.