Does freeze-substitution reveal the hydrophobic nature of interprotofilament bonding in microtubules?

The massive system of microtubules in the ovarioles of Notonecta glauca has been prepared for electron microscopy by means of freeze-substitution. With this technique the protofilaments comprising the microtubule walls can be observed in positive image, indicating that the procedure provides a valuable method for observing microtubule substructure. Surprisingly though, the cryoprotectants, which are an integral part of the method, caused a rearrangement of the microtubules from their usual pattern in the ovarioles. These observations, we believe, provide an insight into intra- and intermicrotubule interactions respectively.     

Geometric optical optimization of the corneal lens of Notonecta glauca

The optimal shape of the corneal lens of the water bug backswimmer (Notonecta glauca) and the optimal shape and position of the thin transition layer between the distal and proximal units of its cornea are theoretically determined. Using a geometric optical method, first the shape of a geometric interface between the lens units is determined, which eliminates the longitudinal spherical aberration. This interface is investigated for differently formed thick lenses when the medium in contact with the entrance surface of the lens is water or air. The optimal transition layer for the amphibious backswimmer is that, the boundaries of which are the theoretical interfaces for water and air, and the refractive index varies continuously in it. The optimal shape of the corneal lens is determined, with the disadvantageous lenses, with respect to the possible minimal spherical aberration and amount of reflected light from the transition layer, being rejected. The optimal position of the transition layer in the cornea can be obtained from the minimization of the amount of diffracted light on the marginal connection of the layers. The optimal corneal lens for backswimmer has ellipsoid boundary surfaces; the optimal transition layer in it is thin bell-shaped, at the marginal connection of which there is no dimple, the maximum of the layer is on the margin of the cornea. The shape of the theoretically optimal corneal lens, the shape and position of the theoretically optimal transition layer agree well with those of Notonecta glauca. The question posed, the geometric optical method used and the results presented are of general importance, and not only with respect to vision in the bug Notonecta, but also in the fossil trilobites, or in the wave guide theories which have been employed in similar modelling problems, in design of system of lenses without spherical aberration, for example.     

Effect of vertebrate and invertebrate kairomones on the life history of Daphnia magna Straus (Crustacea: Branchiopoda)

The history of selection of Daphnia magna populations living in North African temporary ponds may differ from populations inhabiting permanent ponds. Laboratory experiments were conducted to examine the effect of fish Gambusia holbrooki and invertebrate Notonecta glauca kairomones on the life history traits of the freshwater Cladocera Daphnia magna Straus. With fish kairomones, Daphnia reproduced early and had a significantly smaller size at first reproduction (SFR) and a smaller size of neonates compared to control. In contrast, daphnids reared in water treated with Notonecta glauca had no effect on the age at first reproduction but females were also smaller and produced smaller neonates.     

Effects of prey-size and predator-instar on the predation of Daphnia by Notonecta

Abstract. 1. Attack rates and handling times are measured by a series of separate functional response experiments for each instar of Notonecta glauca attacking four size classes of Daphnia magna as prey. The resulting attack rate and handling time surfaces are complex, with maximum attack rates for small predators attacking small prey, and large predators attacking large prey. Adult Notonecta have lower attack rates than the two previous juvenile instars (4 and 5).
2. The literature on attack rates and handling times in other predator—prey interactions that involve a series of different predator and prey size or age classes is reviewed in the context of the Notonecta-Daphnia results. The data suggest that small predator instars will usually compete with large instars for food, unless there is spatial or temporal separation between them.
3. Complex attack rate and handling time surfaces are to be expected wherever a wide range of prey and predator sizes is involved.
4. Size related changes in attack rates and handling times can introduce very complex dynamics into predator-prey interactions.     

Non-interactive multiple predator effects on tadpole survival

Interactions among and within three species of predators were estimated in terms of their effects on prey survival using short-term predation experiments. The prey were tadpoles (Rana temporaria), and the predators were dragonfly larvae (Anax imperator), newts (Triturus alpestris), and backswimmers (Notonecta glauca). Mortality rate per predator imposed by Triturus and Notonecta did not decline with predator density, whereas the predation rate of Anax was strongly reduced when the number of predator individuals increased. Impacts of all three predators were not altered by the presence of other species in pairwise combinations. This system is therefore characterized by interference between individual dragonflies but relatively independent effects of predator species. These results were largely predictable based on the natural history of the predators and are encouraging for attempts to model communities as assemblages of interacting species.     

Mechanisms of cryoprotection in freezing tolerant animal systems

The theoretical mechanisms of freezing protection afforded by the natural occurrence of glycerol in an adult, freezing-tolerant insect have been considered in light of recent findings. While unequivocal identification of the specific site or sites of action of glycerol is yet obscured in a maze of interactions, it is apparent that a multicellular system (organism) that naturally possesses glycerol in high, nonlethal concentrations, that can maintain activity in the presence of tissue ice, that can survive frequent and prolonged freeze-thaw encounters, and finally, that can regulate cryoprotectant levels in the presence of changing environmental conditions should be a major focus of future studies.The carabid beetle, Pterostichus brevicornis has been found to regulate glycerol levels in response to fluctuating ambient temperatures even while frozen. Hemolymph freezing points and whole-body supercooling points correlated well with changes in glycerol. Freezing and supercooling points decreased 0.9 ° C per 4 g/100 ml increase in glycerol.An interpretation of the data accumulated on insect studies and integrated with data from other multicellular systems supports the theory that a single site of freezing concept as applied to considerations of cryoprotection and cryoinjury may not be realistic. Each level of events occurring during the freezing process in glycerolated and nonglycerolated animal systems has been discussed. There appears little room for speculative separation and isolation of the site of action for the events of freezing (and thawing) represent a continuum with changes in each parameter directly dependent upon the entire sequence.     

Insect predation on pike fry

Laboratory tests evaluated the predatory impact of the macroinvertebrates Erythromma najas larvae (Odonata, Zygoptera: Coenagrionidae), Notonecta glauca (Heteroptera: Notonectidae), Ilyocoris cimicoides (Heteroptera: Naucoridae), Libellula depressa larvae (Odonata, Anisoptera: Libellulidae), Dytiscus marginalis larvae (Coleoptera: Dytiscidae) and Anax imperator larvae (Odonata, Anisoptera: Aeshnidae) on 3-, 12-, 21- and 30-day-old pike fry Esox lucius . All these insect predators captured and ate pike fry during the test, although the numbers killed varied among species. Dytiscus marginalis, Anax imperator and Notonecta glauca were the most Voracious predators.     

Ultrastructural cryoinjury and cryoprotection of rough endoplasmic reticulum

One phase of a study on cryosurvival and cryoprotection of mammalian cells, in terms of ultrastructural alteration of rough endoplasmic reticulum (RER) within rat pancreatic acinar cells, is presented. Small (2–3 mm) squares of tissue, 0.7–0.9 mm in thickness, were compared as unfrozen controls, with (w) and without (wo) glycerol pretreatment (15% $\text{v}\text{v}$ in mammalian Ringer's solution) at 0 °C and 22 °C (to regulate glycerol permeability); as well as parallel frozen-thawed samples, after combinations of slow (3.8 °C/min) freezing (SF) and rapid (38 °C/sec) freezing (RF) with either slow (1.5 °C/min) thawing (ST) or rapid (8 °C/sec) thawing (RT). Regimens compared were SFRT, SFST, RFRT, and RFST, all w and wo glycerol pretreatment at 0 °C and 22 °C. Tissue from each treatment was prepared for electron microscopic observations. The results on rates of freezing and thawing and relative cryoprotection of intracellular and extracellular glycerol under conditions described are intended to serve as a correlative basis for subsequent parallel studies on function (protein synthesis) and ultrastructure of the frozen state. They now indicate the following: (1) Cryoinjury of RER, which occurred during all treatments compared, was manifested in irregularity, dilatation, vesiculation, and altered matrix density of cisternae, and ribosomal derangement or disjunction. Least injury was shown by some disorientation and dilatation with increasing degrees of damage involving accentuation of these and other alterations. Such ultrastructural alterations to RER are not unique to cryoinjury, since they have been induced by treatments and agents other than freeze-thawing in experimental pathology. (2) Cryoinjury is unique, however, in that it can be regulated to demonstrate a spectrum of degrees of injury to cells and their organelles, immediately after cryoexposure. Controlled cryoinjury is suggested as a research tool for studies on injury, in general, on an ultrastructural-functional level. (3) Glycerol is injurious or toxic during pretreatment. Toxicity, which resembles cryoinjury, is greater during 22 ° C (intracellular) than 0 °C (extracellular) glycerol pretreatment, especially with respect to dilatation of cisternae. (4) Extra-cellular glycerol is cryoprotective during both slow and rapid freezing followed by either slow or rapid thawing, while little or no cryoprotection is afforded when glycerol is located simultaneously in the intracellular and extracellular location. (5) Rate of freezing is more important than rate of thawing as a factor in cryosurvival. Rapid freezing is more injurious than slow freezing, in the absence of glycerol or in the presence of extracellular glycerol, with slight or no differences seen as a function of thawing rate. Neither rate of freezing nor rate of thawing is of serious consequence when glycerol is intracellular. (6) Rate of thawing has importance after slow freezing, when slow thawing is more injurious than rapid, but not after rapid freezing, either in the presence or absence of extracellular glyeerol.     

High molecular weight microtubule-associated proteins within testes of hemipteran insects.

Results presented here indicate that the high molecular weight microtubule-associated proteins isolated from the ovaries of the hemipterans, Oncopeltus fasciatus and Notonecta glauca, while absent from nervous tissue, are present in the testes of the respective species. Here they are seen to be located to extensive microtubule aggregates within the cells surrounding the sperm, but not obviously to the sperm themselves.     

Partition of synaptic membranes in aqueous two-phase systems at subzero temperatures by using anti-freeze solvent

The freezing point of aqueous two-phase (liquid-liquid) systems containing water, dextran and poly(ethylene glycol) has been lowered by including glycerol. Biological membranes, obtained by fragmentation of a crude synaptosomal preparation from calf brain cortex, have been included in the two-phase systems. The effects of temperature and the concentration of glycerol on the partition of the membranes within the systems have been investigated. Considerable stabilisation of the membranes was noticed when they were partitioned at -10 degrees C compared with 0 degrees C. The influences of glycerol, ethylene glycol, N,N-dimethylformamide and tetrahydrofuran on the phase-forming properties of the systems and on enzyme activities are also presented. Possible use of the above systems for studies and separation of biological membranes are discussed.     

Infective Juveniles of the Entomopathogenic Nematode,Steinernema feltiae Produce Cryoprotectants in Response to Freezing and Cold Acclimation

Steinernema feltiae is a moderately freeze-tolerant entomopathogenic nematode which survives intracellular freezing. We have detected by gas chromatography that infective juveniles of S. feltiae produce cryoprotectants in response to cold acclimation and to freezing. Since the survival of this nematode varies with temperature, we analyzed their cryoprotectant profiles under different acclimation and freezing regimes. The principal cryoprotectants detected were trehalose and glycerol with glucose being the minor component. The amount of cryoprotectants varied with the temperature and duration of exposure. Trehalose was accumulated in higher concentrations when nematodes were acclimated at 5°C for two weeks whereas glycerol level decreased from that of the non-acclimated controls. Nematodes were seeded with a small ice crystal and held at -1°C, a regime that does not produce freezing of the nematodes but their bodies lose water to the surrounding ice (cryoprotective dehydration). This increased the levels of both trehalose and glycerol, with glycerol reaching a higher concentration than trehalose. Nematodes frozen at -3°C, a regime that produces freezing of the nematodes and results in intracellular ice formation, had elevated glycerol levels while trehalose levels did not change. Steinernema feltiae thus has two strategies of cryoprotectant accumulation: one is an acclimation response to low temperature when the body fluids are in a cooled or supercooled state and the infective juveniles produce trehalose before freezing. During this process a portion of the glycerol is converted to trehalose. The second strategy is a rapid response to freezing which induces the production of glycerol but trehalose levels do not change. These low molecular weight compounds are surmised to act as cryoprotectants for this species and to play an important role in its freezing tolerance.     

Changes in feeding behaviour of the larvae of the damselfly Enallagma cyathigerum in response to stimuli from predators

1. The feeding rates, diet composition and diel periodicity in feeding activity among larvae of the damselfly Enallagma cyathigerum Charp., exposed to chemical, visual and mechanical stimuli from the predators Ranatra linearis (L.), Notonecta glauca L. and Rutilus rutilus (L.) were investigated.
2. In response to chemical cues produced by all the predator species, larvae reduced their rate of feeding significantly (especially on large, sedentary prey).
3. Small larvae reacted more strongly than large ones to the kairomone produced by N. glauca.
4. The fish kairomone induced a reduction in feeding activity during daylight hours only. This reaction was more intense than the reaction to non-chemical cues.
5. Observed predator-induced changes in diet composition, caused directly by reduced activity during feeding, are discussed as an antipredator behaviour of damselfly larvae.     

Stabilizing microtubules with taxol increases microfilament stability during freezing of rye root tips

We have used double fluorescence labelling to investigate the effect of freezing on microtubules and microfilaments in root-tip cells of rye (Secale cereale L. cv Rymin). Freezing to -5°C (which does not kill these cells) caused partial depolymerization of both, but microfilaments were more resistant than microtubules. When microtubules were stabilized against freeze-induced depolymerization by pre-treating seedlings with taxol, microfilaments exhibited enhanced stability as well. Almost all the frozen cells containing taxol-stabilized microtubules also contained microfilaments. When seedlings were treated with the microtubule-destabilizing drug APM prior to freezing, microfilaments became more susceptible to freeze-induced depolymerization than in controls. These data suggest a physical interaction between microtubules and microfilaments in these cells.     

The nucleotide-binding state of microtubules modulates kinesin processivity and the ability of Tau to inhibit kinesin-mediated transport

The ability of Tau to act as a potent inhibitor of kinesin's processive run length in vitro suggests that it may actively participate in the regulation of axonal transport in vivo. However, it remains unclear how kinesin-based transport could then proceed effectively in neurons, where Tau is expressed at high levels. One potential explanation is that Tau, a conformationally dynamic protein, has multiple modes of interaction with the microtubule, not all of which inhibit kinesin's processive run length. Previous studies support the hypothesis that Tau has at least two modes of interaction with microtubules, but the mechanisms by which Tau adopts these different conformations and their functional consequences have not been investigated previously. In the present study, we have used single molecule imaging techniques to demonstrate that Tau inhibits kinesin's processive run length in an isoform-dependent manner on GDP-microtubules stabilized with either paclitaxel or glycerol/DMSO but not guanosine-5'-((α,β)-methyleno)triphosphate (GMPCPP)-stabilized microtubules. Furthermore, the order of Tau addition to microtubules before or after polymerization has no effect on the ability of Tau to modulate kinesin motility regardless of the stabilizing agent used. Finally, the processive run length of kinesin is reduced on GMPCPP-microtubules relative to GDP-microtubules, and kinesin's velocity is enhanced in the presence of 4-repeat long Tau but not the 3-repeat short isoform. These results shed new light on the potential role of Tau in the regulation of axonal transport, which is more complex than previously recognized.     

The distribution and function of microtubules in nutritive tubes

The density and distribution of microtubules in the nutritive tubes of three hemipteran insects, Corixa punctata, Notonecta glauca and Dysdercus cingulatus, were analysed from electron micrographs by computer. Both parameters varied amongst all three species, the density of microtubules in Corixa being approximately three times that seen in Dysdercus. The density and distribution of microtubules within the nutritive tubes correlated directly with the size of particle transported by them, suggesting that the microtubules may act as a sieve to preclude certain cellular components from entering the tubes. If microtubules are further involved in the generation of motive force for cytoplasmic transport along the nutritive tubes, this function is not dependent upon the number of microtubules, or upon their arrangement with respect either to each other or to the particle being transported.     

Freezing monolayers of cells without gap junctions

Cells in monolayers have been reported to be more susceptible to freezing injury than the same cell type frozen in dispersed suspensions. There appears to be an enhanced susceptibility to intracellular freezing in the monolayers, which is thought to be facilitated by the presence of gap junctions allowing the spread of ice between neighbouring cells. MDCK Type II cells do not form gap junctions in monolayer culture. When frozen at rates of 0.2 to 10 degrees C/min, monolayers in 10% (v/v) propane-1,2-diol or dimethyl sulphoxide showed little influence of cooling rate on survival. This suggested that, in the absence of gap junctions, cells in monolayers did not display enhanced susceptibility to intracellular freezing. In contrast, however, monolayers frozen in glycerol showed a marked increase in cell damage when cooled at rates higher than 0.5 degrees C/min. This does not necessarily counter the suggestion that lack of gap junctions mitigates intracellular freezing as there is evidence that glycerol may itself promote intracellular freezing.     

The influence of different freezing procedures and different cryoprotective agents on the immunological capacity of frozen-stored lymphocytes

The influence of a series of factors on the frozen storage of lymphocytes was investigated. The cells were frozen using different freezing programmes, using the cryoprotectants dimethyl sulphoxide and glycerol in different concentrations in the freezing medium, and with variations in the period of exposure of cells to cryoprotectants before freezing and after thawing. Cell viability was evaluated by quantitative measurements of the cell-mediated immune response after stimulation with phytohemagglutinin, pokeweed mitogen, concanavalin A, and allogenic cells in mixed lymphocyte cultures. The factors investigated were found to have an important effect on the immune response, so that careful investigation and exact specification of the freezing system are necessary before frozen cells are used in blast-transformation tests. The best freezing programme had a duration of approximately 40 min with a smooth progression through the temperature range where phase transition takes place. The optimum dimethyl sulphoxide concentration in this programme was 8–10%. Dimethyl sulphoxide had no toxic effect on the cells, and no equilibration period was necessary prior to freezing. An equilibration period of 15 min with 10% glycerol was even better than the optimum programme with dimethyl sulphoxide.     

Predation risk perception and food scarcity induce alterations of life-cycle traits of the mosquito Culex pipiens

Abstract.  1. Predators may affect prey populations by direct consumption, and by inducing defensive reactions of prey to the predation risk. Food scarcity frequently has effects on the inducible defences of prey, but no consistent pattern of food–predation risk interaction is known.
2. In this study the combined effect of food shortage and predation-risk perception in larvae of the mosquito Culex pipiens was investigated. Water exposed to the aquatic predator bug Notonecta glauca was used as a source of predation intimidation. Mosquito larvae were reared in three different media containing either no predator cues or the cues of N. glauca that had been fed on either C. pipiens larvae or on Daphnia magna . Food was provided in favourable or limited amount for these set-ups.
3. The results showed that chemical cues from the predators fed with prey's conspecifics caused a decreased survival, delayed pre-imaginal development, and reduction in body size of emerged mosquitoes, whereas chemical cues from predators fed with D. magna caused only delayed development. Food scarcity significantly exacerbates the negative effect of the predator cues on pre-imaginal development of C. pipiens . Effects of the cues on larval development and body size of imagoes are significantly stronger for females than for males.
4. The present study suggests that when food is limited, predators can affect population dynamics of prey not only by direct predation, but also by inducing lethal and sublethal effects due to perception of risk imposed by chemical cues. To understand the effects of predators on mosquito population dynamics, environmental parameters such as food deficiency should be considered.     

Interactions of cooling rate, warming rate, glycerol concentration, and dilution procedure on the viability of frozen-thawed human granulocytes

Difficulties in the successful freezing of human granulocytes could lie at two levels. One is that critical cryobiological variables have not yet been identified, the other is that the inconsistent results may be due to unusual biological aspects of the cell. This paper is concerned with the former. A prerequisite for the successful freezing of mammalian cells is the ability of the cell to tolerate cryoprotective levels of additive. The additive studied here was glycerol. Based on fluorescent staining with fluorescein diacetate, we found that 1 and 2 M concentrations are in fact chemically toxic at 22 degrees C. Superimposed on this toxicity is some osmotic sensitivity to the removal of the additive by other than slow dilution. The dilution procedure was selected on the basis of computer modeling of the osmotic response of the cells. The model requires a value for the permeability coefficient for glycerol. The value (4 X 10(-5) cm/min) was obtained by measuring the rate of increase of the volume of cells in hyperosmotic glycerol. The response of human granulocytes to freezing to -196 degrees C and thawing in 1 or 2 M glycerol was not unusual. The optimum cooling rate was 1-3 degrees C/min, and cooling at 10 degrees C/min or faster was especially deleterious if warming was slow (1 degree C/min) rather than rapid (188 degrees C/min). The FDA assay showed that some 75% of the cells survived freezing and thawing at optimum rates in 1 or 2 M glycerol; and some 50-60% remained viable after the glycerol had been removed, provided that the cells remained at 0 degrees C. However, granulocytes normally function at 37 degrees C. Because chemotaxis is considered a good assay of normal function, we developed a modified procedure capable of discriminating among random migration, enhanced random migration (chemokinesis), and directed cell migration (true chemotaxis). When frozen-thawed-diluted cells were incubated for 60 min at 37 degrees C, their survival, based both on the FDA assay and on the chemotaxis assay, was zero. In fact, a prior exposure of the cells to 2 M glycerol at 0 degrees C, even in the absence of freezing, resulted in a rapid loss in FDA viability when the cells were subsequently held at 37 degrees C for up to 60 min. Survivals based on FDA are usually reported to be considerably higher than survivals based on functional assays such as chemotaxis or phagocytosis.(ABSTRACT TRUNCATED AT 400 WORDS)     

The C-banded karyotypes of the four British species of Notonecta L. (Heteroptera: Notonectidae)

Chromosome preparations were made from mid-gut and ovarian cells of adult Notonecta glauca L., N. obliqua Thunberg, N. maculata F. and N. viridis Delcourt, using the acetic acid dissociation, air-drying method (Crozier 1968) with Giemsa staining. C-banding was obtained by treatment with barium hydroxide and salt-sodium citrate (2xSSC). The karyotypes of the first three species are very similar, with 11 pairs of autosomes plus XY sex chromosomes (plus sometimes a small 12th autosome pair in N. glauca), and the sequence of chromosome sizes very similar. However, the four longest pairs of autosomes, and the X chromosomes, have characteristic C-band patterns, which differ between the species. The karyotype of N. viridis is more distinct, with one pair of long autosomes, while the remaining chromosomes are much shorter. The long autosomes have distinct C-bands, but these are present in only one of the shorter pairs, as faint terminal bands. In warm conditions the long autosomes of N. viridis appear rod-like, but in cold conditions they have one end heavily condensed, giving a tadpole-like appearance.