Behavioural adaptations of Rana temporaria to cold climates

Environmental conditions at the edge of a species’ ecological optimum can exert great ecological or evolutionary pressure at local populations. For ectotherms like amphibians temperature is one of the most important abiotic factors of their environment as it influences directly their metabolism and sets limits to their distribution. Amphibians have evolved three ways to cope with sub-zero temperatures: freeze tolerance, freeze protection, freeze avoidance. The aim of this study was to assess which strategy common frogs at mid and high elevation use to survive and thrive in cold climates. In particular we (1) tested for the presence of physiological freeze protection, (2) evaluated autumnal activity and overwintering behaviour with respect to freeze avoidance and (3) assessed the importance of different high-elevation microhabitats for behavioural thermoregulation. Common frogs did not exhibit any signs of freeze protection when experiencing temperatures around 0 °C. Instead they retreated to open water for protection and overwintering. High elevation common frogs remained active for around the same period of time than their conspecifics at lower elevation. Our results suggest that at mid and high elevation common frogs use freeze avoidance alone to survive temperatures below 0 °C. The availability of warm microhabitats, such as rock or pasture, provides high elevation frogs with the opportunity of behavioural thermoregulation and thus allows them to remain active at temperatures at which common frogs at lower elevation cease activity.     

Avoidance mechanisms of three Pieris butterfly species against the parasitoid wasp Apanteles glomerulus

Abstract. 1. Experimental studies have shown that larvae of three Pieris butterflies, P.rapae L., P.melete Mènètriés and P.napi L., are attacked by a parasitoid wasp, Apanteles glomeratus L. Although P.rapae larvae are parasitized heavily in the field, P.melete and P.napi are infrequently parasitized successfully because they possess mechanisms for encapsulating parasitoid larvae and for avoiding parasitism.
2. This study examines spatial and temporal variation in rates of parasitism of the three Pieris species by A.glomeratus in the field. We attempted to determine whether P.rapae possesses any means of avoiding parasitism by this wasp, and then to deduce why both P.melete and P.napi have more distinctive avoidance mechanisms than P.rapae.
3. Our results indicate that in temporary habitats, which are the main habitats of P.rapae, P.rapae is able to escape A.glomeratus in time and space by colonizing new habitats before the parasitoid arrives. In permanent habitats, however, such escape is not possible. P.rapae larvae lack physiological or behavioural avoidance mechanisms of reducing parasitism rates in permanent habitats. P.melete and P.napi , in contrast, live only in permanent habitats, where the parasitic pressure is potentially high, and have evolved active avoidance mechanisms.     

Evidence of a distribution difference between two gangliosides in bilayer membranes

Freeze-etch electron microscopy, a platinum shadowing technique, has been used to compare the lateral distribution of several gangliosides in bilayer model membranes by directly visualizing bound lectin molecules. In particular, GM₁ and GD_1a, major components of brain ganglioside, were studied in phase-separated mixtures of dipalmitoyl- and dielaidoylphosphatidylcholines exposed to Ricinus communis agglutinin and wheat germ agglutinin. The distribution of glycolipid showed evidence of microheterogeneity in that bound lectin tended to occur in clusters of several or more molecules. With GD_1a as receptor such clusters were small and very uniformly distributed over the membrane surface. Somewhat larger, irregularly spaced clusters of up to a dozen lectin particles were more typical of membranes bearing GM₁ and, in addition, there were occasional extensive patches of bound lectin coexisting with areas apparently devoid of glycolipid receptor in phase-separated mixtures of dipalmitoyl- and dielaidoylphosphatidylcholine. Gangliosides in the latter mixtures were not obviously influenced in their lateral distribution by the presence of coexisting fluid and rigid domains. These basic observations seem to extend to bilayer membranes containing mixtures of two gangliosides. The patterns of lectin binding were not grossly affected by incubation time or history of warming and cooling. This study was extended to bilayers of pure dipalmitoylphosphatidylcholine in expectation that the distinctive features characteristic of the P_β′ phase of this lipid might accentuate any behavioural differences between GM₁ and GD_1a.GM₁ was found to exist preferentially in the ‘trough’ regions between P_β′ ripples, while GD_1a showed no apparent preferential arrangement. Given that bound lectins adequately reflect glycolipid distribution in membranes, it would appear that structurally different glycolipids from the same host membrane can assume different distributions on the basis of interactions with defined lipid host matrices.     

The presence and quantification of splenic ice in the McMurdo Sound Notothenioid fish, Pagothenia borchgrevinki (Boulenger, 1902)

Survival of some polar fishes is associated with high levels of circulating antifreeze glycoproteins (AFGPs). AFGP prevent ice growth giving rise to thermal hysteresis. The inhibiting action of AFGPs implies that polar fish contain ice to which AFGPs adsorb. Cryopelagic Pagothenia borchgrevinki, inhabiting the ice-laden waters of McMurdo Sound, Antarctica, were assayed for ice and ice was found on skin, gills, in the intestine, and in the spleen. Two methods used to assess the number of ice crystals in spleens gave comparable results (12.1 +/− 1.9 and 22 +/− 3.8 per spleen). Attempts were made to measure the rate of uptake of ice by P. borchgrevinki held in cages immediately beneath the sub-ice platelet layer in McMurdo Sound; uptake was sporadic. Introduction of ice into fish by spray freezing a small patch of the integument resulted in detection of splenic ice after 1 h, illustrating that a mechanism exists for ice transport from the periphery to the spleen. Splenic ice did not seem to be eliminated from fish held in ice-free water at − 1.6 °C for approximately two months. The relatively small number of splenic ice crystals and the slow rate of ice uptake suggest efficient ice barriers exist in P. borchgrevinki.     

The ins and outs of water dynamics in cold tolerant soil invertebrates

Many soil invertebrates have physiological characteristics in common with freshwater animals and represent an evolutionary transition from aquatic to terrestrial life forms. Their high cuticular permeability and ability to tolerate large modifications of internal osmolality are of particular importance for their cold tolerance. A number of cold region species that spend some or most of their life-time in soil are in more or less intimate contact with soil ice during overwintering. Unless such species have effective barriers against cuticular water-transport, they have only two options for survival: tolerate internal freezing or dehydrate. The risk of internal ice formation may be substantial due to inoculative freezing and many species rely on freeze-tolerance for overwintering. If freezing does not occur, the desiccating power of external ice will cause the animal to dehydrate until vapor pressure equilibrium between body fluids and external ice has been reached. This cold tolerance mechanism is termed cryoprotective dehydration (CPD) and requires that the animal must be able to tolerate substantial dehydration. Even though CPD is essentially a freeze-avoidance strategy the associated physiological traits are more or less the same as those found in freeze tolerant species. The most well-known are accumulation of compatible osmolytes and molecular chaperones reducing or protecting against the stress caused by cellular dehydration. Environmental moisture levels of the habitat are important for which type of cold tolerance is employed, not only in an evolutionary context, but also within a single population. Some species use CPD under relatively dry conditions, but freeze tolerance when soil moisture is high.     

Gap junctions between guinea-pig pinealocytes

Summary In accordance with previous results in rats, belt-like arrangements of fenestrated gap junctions have been found around the collicular segments of pineal cells in the guinea pig. In addition, macular interpinealocyte gap junctions have been observed in this species.S.-K. Huang was a recipient of a Humboldt Foundation fellowship.     

Use of dynamic mechanical analysis (DMA) to determine critical transition temperatures in frozen biomaterials intended for lyophilization

Dynamic mechanical analysis is widely used to determine glass transitions in solid state materials. However, here we demonstrate the application of DMA for the determination of glass transitions (Tg’) in the frozen liquid state by means of a steel sample pocket. The use of the pocket allows frozen material to be analysed and glass transition events demonstrated. In addition, it allows weak glass transitions to be detected clearly in some complex formulations where they can be obscured by eutectic and other strong thermal events when other methods such as DSC or DTA are used. Classical excipients (trehalose, lactose, dextran) were analysed and shown to give reproducible Tg’ values, though with values slightly higher than those obtained by DSC. Finally, several complex real biological materials, typical of those encountered when freeze drying biological and biopharmaceutical materials, were analysed and the potential value of DMA demonstrated to determine the relevant glass transition temperatures for use in cryobiology and freeze drying.     

Cold resistance in two species of cave-dwelling beetles (Coleoptera: Cholevidae)

Supercooling points (SCPs), lower lethal temperatures (LLTs), and the effect of short-term exposures (1 min) to low temperatures were examined in the adults of two stenothermal leptodirin species, Neobathyscia mancinii and Neobathyscia pasai (Coleoptera, Cholevidae). Specimens were collected from two caves in the Venetian Prealps (NE-Italy). Inter-species comparison highlighted lower values of SCP in N. mancinii (−7.1±0.9 °C) than in N. pasai (−6.4±0.3 °C), with no significant intersexual differences in both species. N. pasai (LLT₅₀±SE=−16.96±2.30 °C; LLT₁₀₀=−25.41 °C) tolerated short exposures to subzero temperatures better than N. mancinii (LLT₅₀±SE=−4.89±1.08 °C; LLT₁₀₀=−11.72 °C). According to the mortality and cumulative proportion of individual freezing curves (CPIF), SCPs and LLT₁₀₀, N. pasai may be defined as “strongly freeze tolerant”, N. mancinii as “moderately freezing tolerant”. Overall, these results may justify the different in-cave habitat selection showed by the two species (N. pasai was abundant close to the entrance where the temperature is variable whereas N. mancinii was confined to the internal part of the cave where the temperature is constant throughout the year), and suggest hypotheses on the effects of such habitat selection on freeze tolerance strategy adopted. Finally, they give new insights into possible responses to climate changes in cave dwelling species.     

Evaluation and preservation of vascular architectures in decellularized whole rat kidneys

Organ transplantation is the gold standard treatment for end-stage organ failure. Due to the severe shortage of transplantable organs, only a tiny fraction of patients may receive timely organ transplantation every year. Decellularization-recellularization technology using allogeneic and xenogeneic organs is currently conceived to be a promising solution to generate functionally transplantable organs in vitro. This approach, however, still faces tremendous technological challenges, one of them being the ability to evaluate and preserve the integrity of vascular architectures upon decellularization and cryostorage of the whole organ matrices so that the off-the-shelf organ grafts are available on demand for clinical applications. In the present study, we report a Micro-CT imaging method for evaluating the integrity of vasculature of the decellularized whole organ scaffolds with/without freezing/thawing. The method uses radiopaque Microfil perfusion and x-ray fluoroscopy to acquire high-resolution angiography of the organ matrix. The whole rat kidney is decellularized using a new multistep perfusion protocol with the combined use of Triton X-100 and DNase. The decellularized kidney matrix is then cryopreserved after the pretreatment with different cryoprotectant solutions. The reconstructed tomographic images from Micro-CT confirm various structural alterations in the vasculature of the whole decellularized kidney matrix with/without frozen storage. The freezing damage to the vascular architectures can be reduced by perfusing cryoprotectant solutions into the whole kidney matrix. Ice-free cryopreservation with the vitrification solution VS83 can successfully preserve the integrity of the whole kidney matrix's vasculature after frozen storage.     

High subzero cryofixation: A technique for observing ice within tissues

While various fixation techniques for observing ice within tissues stored at high sub-zero temperatures currently exist, these techniques require either different fixative solution compositions when assessing different storage temperatures or alteration of the sample temperature to enable alcohol-water substitution. Therefore, high-subzero cryofixation (HSC), was developed to facilitate fixation at any temperature above −80 °C without sample temperature alteration. Rat liver sections (1 cm²) were frozen at a rate of −1 °C/min to −20 °C, stored for 1 h at −20 °C, and processed using classical freeze-substitution (FS) or HSC. FS samples were plunged in liquid nitrogen and held for 1 h before transfer to −80 °C methanol. After 1, 3, or 5 days of −80 °C storage, samples were placed in 3% glutaraldehyde on dry ice and allowed to sublimate. HSC samples were stored in HSC fixative at −20 °C for 1, 3, or 5 days prior to transfer to 4 °C. Tissue sections were paraffin embedded, sliced, and stained prior to quantification of ice size. HSC fixative permeation was linear with time and could be mathematically modelled to determine duration of fixation required for a given tissue depth. Ice grain size within the inner regions of 5 d samples was consistent between HSC and FS processing (p = 0.76); however, FS processing resulted in greater ice grains in the outer region of tissue. This differed significantly from HSC outer regions (p = 0.016) and FS inner regions (p = 0.038). No difference in ice size was observed between HSC inner and outer regions (p = 0.42). This work demonstrates that HSC can be utilized to observe ice formed within liver tissue stored at −20 °C. Unlike isothermal freeze fixation and freeze substitution alternatives, the low melting point of the HSC fixative enables its use at a variety of temperatures without alteration of sample temperature or fixative composition.