Effects of cold narcosis on memory acquisition,consolidation and retrieval in honeybees(Apis mellifera)

In learning and memory studies on honeybees(Apis mellifera),cold-induced narcosis has been widely used to temporarily immobilize honeybees.In this study,we investigated the effects of cold narcosis on the associative memories in honeybees by using the proboscis extension response(PER)paradigm.Severe impairments in memory acquisition was found when cold narcosis was performed 30 min,instead of 1 h before training.Locomotor activities were reduced when honeybees were tested 15 min,instead of30 min after cold narcosis.These results indicate that cold narcosis impairs locomotor activities,as well as memory acquisition in a time-dependent manner,but by comparison no such effects on memory retrieval have yet been observed.[0]     

Behavioral studies on tarsal gustation in honeybees: sucrose responsiveness and sucrose-mediated olfactory conditioning

Although the forelegs of honeybees are one of their main gustatory appendages, tarsal gustation in bees has never been systematically studied. To provide a more extensive account on honeybee tarsal gustation, we performed a series of behavioral experiments aimed at characterizing (1) tarsal sucrose sensitivity under different experimental conditions and (2) the capacity of tarsal sucrose stimulation to support olfactory conditioning. We quantified the proboscis extension reflex to tarsal sucrose stimulation and to odors paired with tarsal sucrose stimulation, respectively. Our experiments show that tarsal sucrose sensitivity is lower than antennal sucrose sensitivity and can be increased by starvation time. In contrast, antennae amputation decreases tarsal sucrose sensitivity. Furthermore, we show that tarsal sucrose stimulation can support olfactory learning and memory even if the acquisition level reached is relatively low (40%).     

Effects of sequential exposures of sub-lethal doses of amitraz and thiacloprid on learning and memory of honey bee foragers,Apis mellifera

Pollinators, honey bees in particular, are continuously exposed to various mixtures of pesticides, which contribute to their population decline. Both amitraz and thiacloprid have been proven less toxic to honey bees and are frequently applied in- and out-hive, respectively. We examined the sub-lethal effects of amitraz, thiacloprid and their sequential exposure on learning, memory and sugar responsiveness in Apis mellifera using the Proboscis extension response (PER). Sub-lethal doses of amitraz (0.1, 0.2 and 0.4 µg/bee) and thiacloprid (0.05, 0.1 and 0.2 µg/bee) were tested. Sub-lethal effects were observed only at the highest doses of each pesticide treatment; amitraz (0.4 µg/bee) and thiacloprid (0.2 µg/bee) but not in lower doses. In sequential treatment of amitraz and thiacloprid, reduced acquisition and memory retention were significant across all tested doses. The same profile was also obtained on sugar responsiveness of foragers. Our results suggest that the sequential exposure would pose higher risk to honey bee compared to single pesticide exposure by reducing the bees’ appetitive olfactory learning, memory and sugar acuity more than individual pesticide exposures.     

Effects of gibberellic acid on sucrose accumulation and sucrose biosynthesizing enzymes activity during banana ripening

During banana ripening there is a massive conversion into sugars, mainly sucrose, which can account for more than 10% of the fresh weight of the fruit. An ethylene burst is the trigger of the banana ripening process but there is evidence that other compounds can act as modulators of some biochemical pathways. As previously demonstrated, gibberellic acid (GA₃) can impair the onset of starch degradation and affect some degradative enzymes, but effects on the sucrose biosynthetic apparatus have not yet been elucidated. Here, the activity and amount of sucrose synthase (SuSy; E.C. 2.4.1.13) and sucrose–phosphate synthase (SPS; E.C. 2.4.1.14), respiration rates, ethylene production, and carbohydrate levels, were evaluated in GA₃-infiltrated and non-infiltrated banana slices. The exogenous supply of gibberellin did not alter the respiration or the ethylene profile but delayed sucrose accumulation by at least 2 days. While SuSy activity was similar in control and treated slices, SPS increase and sucrose accumulation was related in treated slices. Western blotting with specific antiserum showed no apparent effects of GA₃ on the amount of SuSy protein, but impaired the increase in SPS protein during ripening. The overall results indicate that although GA₃ did not block carbohydrate mobilisation in a irreversibly way, it clearly affected the triggering of starch breakdown and sucrose synthesis. Also, the delayed sucrose accumulation in GA₃-infiltrated slices could be explained by the disturbance of SPS activity. In conclusion, gibberellins can play an important role during banana ripening and our results also reinforce the idea of multiple regulatory components in the ripening pathway, as evidenced by the GA₃ effects.     

Responsiveness to sucrose and habituation of the proboscis extension response in honey bees

In honey bees, complex behaviours such as associative learning correlate with responsiveness to sucrose. In these behaviours, the subjective evaluation of a sucrose stimulus influences the behavioural performance. Habituation is a well-known form of non-associative learning. In bees, the proboscis extension response can be habituated by repeatedly stimulating the antennae with a low sucrose concentration. A high sucrose concentration can dishabituate the response. This study tests whether habituation correlates with responsiveness to sucrose in bees of different behavioural states and in bees which are habituated with different sucrose concentrations. Habituation and dishabituation in newly emerged bees, 5-day-old bees and foragers strongly correlated with responsiveness to sucrose. Bees with high responsiveness to sucrose displayed a lower degree of habituation and showed greater dishabituation than bees with low responsiveness. The degree of habituation and dishabituation also depended on the concentration of the habituation stimulus. These experiments demonstrate for the first time in a non-associative learning paradigm that the subjective strength of a sucrose stimulus determines the behavioural performance. Non-associative learning shares this property with associative learning, which suggests that the two processes might rely on similar neural mechanisms.Abbreviations: GRS Gustatory response score - PER Proboscis extension response     

Stimulus magnitude effects on the extinction of conditioned consummatory responses to flavored sucrose solution in mice

Paradoxical extinction effects in the conditioned consummatory behavior of rodents have remained largely elusive. Here, appetitive flavor conditioning was studied to determine if a paradoxical magnitude of reinforcement extinction effect (MREE) can occur in the consummatory behavior of mice. During acquisition training of two experiments with factorial design, animals received daily access to either 32% or 4% sucrose solution, and goal tracking time was measured in one-minute bins. In Experiment 1 the solutions were flavored with either 5% or 0.5% almond essence and in Experiment 2 with 2% almond essence, but combined with continuous or partial schedules of reinforcement. During extinction tests of Experiment 1 and 2, water flavored with 0.5% or 2% almond was presented, respectively. Consummatory performance decreased more abruptly during the initial portion of the extinction sessions after training with 32% as compared to 4% sucrose solution. Furthermore, when given a choice test after extinction training (Experiment 2), animals trained with 32% sucrose, preferred the flavored solution, but animals trained with 4% preferred the unflavored solution. These results are interpreted as indicative of the occurrence of a paradoxical MREE in conditioned consummatory behaviors.     

Physiological and morphological characterization of honeybee olfactory neurons combining electrophysiology,calcium imaging and confocal microscopy

The insect antennal lobe is the first brain structure to process olfactory information. Like the vertebrate olfactory bulb the antennal lobe is substructured in olfactory glomeruli. In insects, glomeruli can be morphologically identified, and have characteristic olfactory response profiles. Local neurons interconnect glomeruli, and output (projection) neurons project to higher-order brain centres. The relationship between their elaborate morphology and their physiology is not understood. We recorded electrophysiologically from antennal lobe neurons, and iontophoretically injected a calcium-sensitive dye. We then measured their spatio-temporal calcium responses to a variety of odours. Finally, we confocally reconstructed the neurons, and identified the innervated glomeruli. An increase or decrease in spiking frequency corresponded to an intracellular calcium increase or decrease in the cell. While intracellular recordings generally lasted between 10 and 30 min, calcium imaging was stable for up to 2 h, allowing a more detailed physiological analysis. The responses indicate that heterogeneous local neurons get input in the glomerulus in which they branch most strongly. In many cases, the physiological response properties of the cells corresponded to the known response profile of the innervated glomerulus. In other words, the large variety of response profiles generally found when comparing antennal lobe neurons is reduced to a more predictable response profile when the innervated glomerulus is known.Abbreviations ACT antenno-cerebralis-tract - AL antennal lobe - AP action potential - l-ACT lateral ACT - LN local neuron - LPL lateral protocerebral lobe - m-ACT medial ACT - MB mushroom body - OSN olfactory sensory neuron - PN projection neuron - T1 tract 1 of the antennal nerve     

Sucrose acceptance,discrimination and proboscis responses of honey bees (<Emphasis Type="Italic">Apis mellifera</Emphasis> L.) in the field and the laboratory

Laboratory studies in honey bees have shown positive correlations between sucrose responsiveness, division of labour and learning. We tested the relationships between sucrose acceptance and discrimination in the field and responsiveness in the laboratory. Based on acceptance in the field three groups of bees were differentiated: (1) bees that accept sucrose concentrations >10%, (2) bees that accept some but not all of the sucrose concentrations <10% and water, and (3) bees that accept water and all offered sucrose concentrations. Sucrose acceptance can be described in a model in which sucrose- and water-dependent responses interact additively. Responsiveness to sucrose was tested in the same bees in the laboratory by measuring the proboscis extension response (PER). The experiments demonstrated that PER responsiveness is lower than acceptance in the field and that it is not possible to infer from the PER measurements in the laboratory those concentrations the respective bees accepted in the field. Discrimination between sucrose concentrations was tested in three groups of free-flying bees collecting low, intermediate or high concentrations of sucrose. The experiments demonstrated that bees can discriminate between concentrations differences down to 0.2 relative log units. There exist only partial correlations between discrimination, acceptance and PER responsiveness.     

Expression,purification and immobilization of the intracellular invertase INVA,from <Emphasis Type="Italic">Zymomonas mobilis</Emphasis> on crystalline cellulose and Nylon-6

This paper presents two immobilization methods for the intracellular invertase (INVA), from Zymomonas mobilis. In the first method, a chimeric protein containing the invertase INVA, fused through its C-terminus to CBD _Cex from Cellulomonas fimi was expressed in Escherichia coli strain BL21 (DE3). INVA was purified and immobilized on crystalline cellulose (Avicel) by means of affinity, in a single step. No changes were detected in optimal pH and temperature when INVA-CBD was immobilized on Avicel, where values of 5.5 and 30 °C, respectively, were registered. The kinetic parameters of the INVA-CBD fusion protein were determined in both its free form and when immobilized on Avicel. K _m and V _max were affected with immobilization, since both showed an increase of up to threefold. Additionally, we found that subsequent to immobilization, the INVA-CBD fusion protein was 39% more susceptible to substrate inhibition than INVA-CBD in its free form. The second method of immobilization was achieved by the expression of a 6xHis-tagged invertase purified on Ni-NTA resin, which was then immobilized on Nylon-6 by covalent binding. An optimal pH of 5.5 and a temperature of 30 °C were maintained, subsequent to immobilization on Nylon-6 as well as with immobilization on crystalline cellulose. The kinetic parameters relating to V _max increased up to 5.7-fold, following immobilization, whereas K _m increased up to 1.7-fold. The two methods were compared showing that when invertase was immobilized on Nylon-6, its activity was 1.9 times that when immobilized on cellulose for substrate concentrations ranging from 30 to 390 mM of sucrose.     

Effects of stressors in adolescence on learning and memory in rodent models

This article is part of a Special Issue "Puberty and Adolescence".     

Effect of pheromones,hormones, and handling on sucrose response thresholds of honey bees (Apis mellifera L.)

The responsiveness of bees to sucrose is an important indicator of honey bee foraging decisions. Correlated with sucrose responsiveness is forage choice behavior, age of first foraging, and conditioned learning response. Pheromones and hormones are significant components in social insect systems associated with the regulation of colony-level and individual foraging behavior. Bees were treated to different exposure regimes of queen and brood pheromones and their sucrose responsiveness measured. Bees reared with queen or brood pheromone were less responsive than controls. Our results suggest responsiveness to sucrose is a physiologically, neuronally mediated response. Orally administered octopamine significantly reduced sucrose response thresholds. Change in response to octopamine was on a time scale of minutes. The greatest separation between octopamine treated and control bees occurred 30 min after feeding. There was no significant sucrose response difference to doses ranging from 0.2 g to 20 g of octopamine. Topically applied methoprene significantly increased sucrose responsiveness. Handling method significantly affected sucrose responsiveness. Bees that were anesthetized by chilling or CO₂ treatment were significantly more responsive than control bees 30 min after handling. Sixty minutes after handling there were no significant treatment differences. We concluded that putative stress effects of handling were blocked by anesthetic.Abbreviations BP brood pheromone - JH juvenile hormone - OA octopamine - PER proboscis extension response - PER-RT proboscis extension response threshold - QMP queen mandibular pheromone     

Effect of ovary preservation period on recovery rate and categories of dromedary camel oocytes

The aim of this study was to evaluate the effect of different periods of ovary preservation at 25–30 °C for 5, 6, 7, 9, 12 and 24 h on recovery rate and oocyte categories of dromedary camel oocytes. Camel ovaries were collected from El-Bassatein slaughterhouse, Cairo. The collected ovaries were placed immediately after slaughtering into thermos in saline solution (0.9% NaCl) supplemented with antibiotics (100 IU penicillin and 100 μg streptomycin/ml) at 25–30 °C and transported to the laboratory within 4–5 h. Ovaries were washed three times with warmed (30 °C) phosphate buffer solution (PBS) and one time with ethanol (70%). All visible follicles on the ovarian surface (2–8 mm in diameter) were counted. Oocytes were aspirated using a 20-gauge hypodermic needle. Oocyte yield was recorded and the number of oocytes/ovary was calculated. Oocytes were classified into five categories (compact, partial denuded, denuded, shrunken and cleaved oocytes). Results show that average number of follicles on each ovary was not significantly affected by preservation period, although tended to reduce only after 5 h of ovary preservation. However, this number was insignificantly reduced by increasing period of ovary preservation more than 5 up to 24 h. Average number of oocytes on each ovary was significantly (P < 0.05) reduced only between 5 and 6 h of ovary preservation. Average number of oocytes showed higher reduction rate between 5 and 6 h from 12.4 to 9.3/ovary as well as between 9 and 12 h. Oocyte recovery rate showed insignificant decrease from 88.1% at 5 h to 78.6% at 9 h of preservation. However, it showed significant (P < 0.05) reduction to 62.0% between 9 and 12 h, then insignificantly decreased to 58.6 at 24 h of preservation of the ovaries. Frequency distribution and recovery rate of each category was the highest for compact oocytes and the lowest for cleaved oocytes at all periods of preservation. Increasing preservation period significantly (P < 0.05) decreased frequency distribution of compact and cleaved oocytes, while increased frequency distribution of partial denuded, denude and shrunken oocytes.It might be concluded from the present results that the preservation of dromedary camel ovaries at 25–30 °C for 5–6 h was effective for maintaining the oocytes quality and recovery rate compared with the other preservation periods.     

Effects of non-frozen cold storage on the growth,organogenesis and secondary metabolism of callus cultures

Callus tissues derived from chilling-tolerant herbaceous plant, Atractylodes lancea, Atropa belladonna, Bupleurum falcatum, Dioscorea tokoro, Lithospermum erythrorhizon and Phytolacca americana could be cold-stored at 4°C for three months or more, whereas those from chilling-sensitive herbaceous plants such as Datura innoxia and Perilla frutescens var. crispa and a deciduous tree, Mallotus japonicus, could not survive after cold storage for two to three months. Tobacco callus cultures could be stored at 4°C for two or four months depending on a callus strain. The effect of cold storage on secondary metabolite production varied. Nicotine and betalain production suffered from cold storage of tobacco and Phytolacca americana callus cultures, respectively. However, production of anthocyanin in cultures of Mallotus japonicus and Bupleurum falcatum and shikonin derivatives in Lithospermum erythrorhizon callus was affected very little. Root-forming ability was retained for more than one year in cold-stored callus tissues of Bupleurum falcatum, while the control callus tissues maintained at 25°C completely lost the organogenetic ability six months after the first subculture.     

Effects of cold stress on glutathione and related enzymes in rat erythrocytes

Effects of acute and chronic cold stress on glutathione and related enzymes in rat erythrocytes were investigated. Blood from both cold-acclimated (CA) and cold-adapted (CG) rats had significantly lower concentrations of glutathione than blood from control animals. Superoxide dismutase activity was increased significantly in CA rats and tended to rise in CG rats. Activity of glutathione peroxidase in erythrocytes was inconsistent in that it tended to increase in CA rats but decreased significantly in CG rats. The results may imply that CG rats suffered deleterious effects of hydrogen peroxide. On the other hand, there were marked decreases in glutathione peroxidase and glutathione reductase activities in acutely cold-exposed rats in conjunction with unchanged levels of glutathione. In all treatments the state of riboflavin metabolism was estimated to be adequate, since no increases were observed in the erythrocyte glutathione reductase activity coefficient.     

Effects of cold, starvation and immobilization on the composition of acid-soluble nuclear proteins in chicken liver

Nuclear proteins soluble in 0.2 M sulphuric acid were isolated from the liver of three groups of hens subjected for 60 hours to starvation, immobilization or cold exposure. The obtained proteins were separated by means of one-dimensional and two-dimensional electrophoresis on polyacrylamide gel. It was observed that this exposure of the birds to stress caused no qualitative changes in liver nuclear proteins. Histones, histone-like proteins - M1, M2, M3, uM1, HMG 1 and 2 proteins, and a large group of non-histone protein fractions gave nearly identical patterns. However, several components of nuclear proteins were found whose quantity changed in the liver of the birds subjected to stress. These changes were observed in a protein with molecular weight about 27 000 daltons and two proteins weighing over 100 000 daltons.     

Effects of cold exposure on cyclic AMP concentration in plasma,liver, and brown and white adipose tissues in cold-acclimated rats

Effects of acute cold exposure on plasma energy substrates and tissue 3,5-adenosine monophosphate (cAMP) were analyzed in intact rats, to define an involvement of the nucleotide in nonshivering thermogenesis (NST) and resultant cold acclimation. After an acute cold exposure to –5°C, the plasma glucose level increased gradually in warm-kept control rats (C) while it decreased significantly in cold-acclimated rats (CA). However, it was increased considerably by an extreme cold exposure to –15°C in both C and CA. By contrast, plasma levels of free fatty acids (FFA) increased immediately after cold exposure and the release lasted during the period of exposure especially in C. The cold exposure also increased plasma cAMP concentration but no concomitant increase was found in the liver. In both brown (IBAT) and white (WAT) adipose tissues the nucleotide concentration showed a stepwise decrease. The observed correlation between lipolysis and plasma cAMP response after cold exposure suggests an involvement of the adenylate cyclase-cAMP system in NST via lipid metabolism, at least, in the early stages of cold acclimation.Abbreviations cAMP cyclic 3,5-adenosine monophosphate - NST nonshivering thermogenesis - FFA free fatty acids - IBAT brown adipose tissue - WAT white adipose tissue     

Effects of salicylic acid and cold on freezing tolerance in winter wheat leaves

The effects of salicylic acid (SA) (0.01, 0.1 and 1 mM) and cold on freezing tolerance (freezing injury and ice nucleation activity) were investigated in winter wheat (Triticum aestivum cv. Dogu-88) grown under control (20/18 °C for 15, 30 and 45-day) and cold (15/10 °C for 15-day, 10/5 °C for 30-day and 5/3 °C for 45-day) conditions. Cold acclimatisation caused a decrease of injury to leaf segments removed from the plants and subjected to freezing conditions. Exogenous SA also decreased freezing injury in the leaves grown under cold (15/10 °C) and control (15 and 30-day) conditions. Cold conditions (10/5 and 5/3 °C) caused an increase in ice nucleation activity by apoplastic proteins, which were isolated from the leaves. For the first time, it was shown that exogenous SA caused an increase in ice nucleation activity under cold (15/10 and 10/5 °C) and control conditions. These results show that salicylic acid can increase freezing tolerance in winter wheat leaves by affecting apoplastic proteins.     

Effects of cold exposure and shear stress on endothelial nitric oxide synthase activation

Endothelial nitric oxide synthase (eNOS) is the primary enzyme that produces nitric oxide (NO), which plays an important role in blood vessel relaxation. eNOS activation is stimulated by various mechanical forces, such as shear stress. Several studies have shown that local cooling of the human finger causes strong vasoconstriction, followed after several minutes by cold-induced vasodilation (CIVD). However, the role played by endothelial cells (ECs) in blood vessel regulation in respond to cold temperatures is not fully understood. In this study, we found that low temperature alone does not significantly increase or decrease eNOS activation in ECs. We further found that the combination of shear stress with temperature change leads to a significant increase in eNOS activation at 37 °C and 28 °C, and a decrease at 4 °C. These results show that ECs play an important role in blood vessel regulation under shear stress and low temperature.