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An important objective of wheat improvement programmes is to breed varieties for high yield in optimum conditions and for minimum yield reduction under stress-prone conditions such as heat and drought. Analyses of yield and its components in multiple years allow a comprehensive and comparative understanding of genetic yield potential and stress-tolerance mechanisms in the study germplasm. The present study was carried out to evaluate performance of elite varieties and landraces of spring durum wheat under different water regimes, determine the repeatability of the examined traits, and identify superior genotypes for their potential use in breeding for drought tolerance. A total of 97 accessions of spring durum wheat (T. durum Desf.) were evaluated under rain-fed and well-watered conditions in the nursery of the Centre for Agricultural Research at Martonvásár, Hungary (2011–2013). The experiments were laid out in an unbalanced, incomplete alpha lattice block design. The trait with the lowest broad-sense repeatability was seed length (0.075), while high h2 values were observed for heading date (0.89), thousand-grain weight (0.85) and the protein content (0.85). Grain yield showed moderate level of repeatability (0.53) across the three years. The principal component analysis revealed that grain yield (t/ha) is positively associated with the fertile tiller number, chlorophyll content values at early waxy ripeness stages and plant height. Based on biplot analysis, ‘DP-133′, ‘DP-017′ and ‘DP-061′ proved to be the best durum cultivars in terms of yield whereas genotypes ‘DP-011′, ‘DP-185′, 'DP-126′ and 'DP-136′ preceded them with their good yield stability.

Light spectra influence barley development, causing a diverse range of responses among cultivars that are poorly understood. Here, we exposed three barley genotypes with different light sensitivities to two light sources: fluorescent bulbs, over-representing green and red wavebands, and metal halide lamps, with a more balanced spectrum. We used RNA sequencing to identify the main genes and pathways involved in the different responses, and RT-qPCR to validate the expression values. Different grades of sensitivity to light spectra were associated with transcriptional reprogramming, plastid signals, and photosynthesis. The genotypes were especially divergent in the expression of genes regulated by transcription factors from MADS-box, WRKY, and NAC families, and in specific photoreceptors such as phytochromes and cryptochromes. Variations in light spectra also affected the expression of circadian clock, flowering time, and frost tolerance genes, among others, resembling plant responses to temperature. The relation between PPD-H1, HvVRN1, and HvFT1 expression might explain genotypic differences. Light-sensitive genotypes experienced a partial reversion of the vernalization process and senescence-related stress under the less favorable light quality conditions. The observed light-quality sensitivities reveal a complex mechanism of adaptation to regions with specific light quality features and/or possible regulation of light spectra in plant development during early spring.HighlightDevelopment genes were affected by light quality in the barley varieties tested. Different grades of sensitivity were related to the expression of transcription factors, senescence, light signaling and cold-regulated genes.

The taxonomy of genera Anabaena and Nostoc is very controversial. They are typically paraphyletic within phylogenetic trees and show similar morphological characters. The present study aimed to determine the taxonomic relationships among Anabaena and Nostoc strains of the Mosonmagyarovar Algal Culture Collection (MACC) using 16S rRNA and rbcLX gene sequences. We concluded on the basis of the number of unsuccessful amplifications that more of the examined MACC Nostoc cultures are axenic than the Anabaena cultures. In agreement with previous studies we noticed that the applied phylogenetic algorithms gave congruent results in phylogenetic analyses. However, the genus Nostoc clearly was found not monophyletic in the present study and this finding differed from many of the previous studies. Molecular results contradicted the previous morphology-based classification of some MACC cyanobacteria strains, therefore polyphasic taxonomic approaches are required for the reliable identification of cyanobacterial species. Some strains seemed to be identical based on the alignment of 16S rRNA or rbcLX sequences.

With a possible reference to heat priming and to characterize the extent and variation in the heat stress responses in wheat, the effects of single vs. repeated heat stresses were examined by measuring the changes in morphological and grain yield-related traits and photosynthetic parameters. To achieve these objectives, 51 winter wheat cultivars of various geographic origins were included in two independent experiments covering different phenological stages. In Experiment I, a single heat stress event was applied at stem elongation (SE) and booting (B), and the repeated heat stress was applied at both of these stages (SE+B). In Experiment II, the single heat stress was applied at stem elongation (SE) and full heading (CH), while the repeated heat stress was applied at both stages (SE+CH). While genotype was a more important factor for determining the morphological and yield-related traits, it was the treatment effect that mostly influenced the photosynthetic parameters, with the exception of the chlorophyll content. The heading stage was more sensitive to heat stress than the booting stage, which was primarily due to the larger decrease in the average seed number. The importance of biomass in contributing to grain yield intensified with the heat stress treatments. There was a large variation between the wheat cultivars not only in yielding abilities under control conditions but also in sensitivities to the various heat stresses, based on which 7 distinct groups with specific response profiles could be identified at a highly significant level. The 7 wheat groups were also characterized by their reaction patterns of different magnitudes and directions in their responses to single vs. repeated heat stresses, which depended on the phenological phases during the second cycle of heat stress. The possible association between these findings and heat priming is discussed.

Background Plants use light wavelength, intensity, direction and duration to predict imminent seasonal changes and to determine when to initiate physiological and developmental processes. Among them, crop responses to light are not fully understood. Here, we study how light quality affects barley development, using two broad-spectrum light sources, metal halide (M) and fluorescent (F) lamps. Eleven varieties with known allelic variants for the major flowering time genes were evaluated under controlled conditions (long days, same light intensity). Two experiments were carried out with fully-vernalized plants: 1) control treatments (M, F); 2) shifting chambers 10 days after the start of the experiment (MF, FM). Results In general, varieties developed faster under longer exposure to M conditions. The greatest differences were due to a delay promoted by F light bulbs, especially in the time to first node appearance and until the onset of stem elongation. Yield related-traits as the number of seeds were also affected by the conditions experienced. However, not each variety responded equally, and they could be classified in insensitive and sensitive to light quality. Expression levels of flowering time genes HvVRN1, HvFT1 and PPD-H1 were high in M, while HvFT3 and HvVRN2 were higher under F conditions. The expression under shift treatments revealed also a high correlation between HvVRN1 and PPD-H1 transcript levels. Conclusions The characterization of light quality effects has highlighted the important influence of the spectrum on early developmental stages, affecting the moment of onset of stem elongation, and further consequences on the morphology of the plant and yield components. We suggest that light spectra control the vernalization and photoperiod genes probably through the regulation of upstream elements of signalling pathways. The players behind the different responses to light spectra found deserve further research, which could help to optimize breeding strategies.

The yield potential of wheat depends not only on genetic × environmental interactions, but also on various agronomic factors such as sowing date or the seed rate used for sowing. The main aim of this work was to determine possible correlations between the effects of different sowing dates and plant densities on the yield components of a collection of 48 wheat genotypes. Two-way analysis of variance on the data revealed that both sowing date and plant density, as main components, only had a minor effect on the yield component patterns. Correlation analysis, however, indicated that the sowing date had a greater effect on the yield components, while plant density was in closer correlation with the heading time (r = 0.90). The patterns determined for individual yield components at two different sowing dates and plant densities showed significant differences for spike length, spike fertility, grain number in the main spike, number of productive tillers, grain number on side tillers, mean grain number and grain weight. Genotypes that carry the winter (recessive) alleles of genes regulating vernalisation processes (VRN-A1, VRN-B1, VRN-D1) and the sensitive (recessive) alleles of the two genes responsible for photoperiod sensitivity (PPD-B1, PPD-D1) may have better tillering and consequently higher grain yield, though this may depend greatly on the year.

In addition to its role in vernalization, temperature is an important environmental stimulus in determining plant growth and development. We used factorial combinations of two photoperiods (16H, 12H) and three temperature levels (11, 18 and 25 °C) to study the temperature responses of 19 wheat cultivars with established genetic relationships. Temperature produced more significant effects on plant development than photoperiod, with strong genotypic components. Wheat genotypes with PPD-D1 photoperiod sensitive allele were sensitive to temperature; their development was delayed by higher temperature, which intensified under non-inductive conditions. The effect of temperature on plant development was not proportional; it influenced the stem elongation to the largest extent, and warmer temperature lengthened the lag phase between the detection of first node and the beginning of intensive stem elongation. The gene expression patterns of VRN1, VRN2 and PPD1 were also significantly modified by temperature, while VRN3 was more chronologically regulated. The associations between VRN1 and VRN3 gene expression with early apex development were significant in all treatments but were only significant for later plant developmental phases under optimal conditions (16H and 18 °C). Under 16H, the magnitude of the transient peak expression of VRN2 observed at 18 and 25 °C associated with the later developmental phases.

Pituitary adenylate cyclase-activating polypeptide (PACAP) rescues dopaminergic neurons from neurodegeneration and improves motor changes induced by 6-hydroxy-dopamine (6-OHDA) in rat parkinsonian models. Recently, we investigated the molecular background of the neuroprotective effect of PACAP in dopamine (DA)-based neurodegeneration using rotenone-induced snail and 6-OHDA-induced rat models of Parkinson's disease. Behavioural activity, monoamine (DA and serotonin), metabolic enzyme (S-COMT, MB-COMT and MAO-B) and PARK7 protein concentrations were measured before and after PACAP treatment in both models. Locomotion and feeding activity were decreased in rotenone-treated snails, which corresponded well to findings obtained in 6-OHDA-induced rat experiments. PACAP was able to prevent the behavioural malfunctions caused by the toxins. Monoamine levels decreased in both models and the decreased DA level induced by toxins was attenuated by ∼50% in the PACAP-treated animals. In contrast, PACAP had no effect on the decreased serotonin (5HT) levels. S-COMT metabolic enzyme was also reduced but a protective effect of PACAP was not observed in either of the models. Following toxin treatment, a significant increase in MB-COMT was observed in both models and was restored to normal levels by PACAP. A decrease in PARK7 was also observed in both toxin-induced models; however, PACAP had a beneficial effect only on 6-OHDA-treated animals. The neuroprotective effect of PACAP in different animal models of Parkinson's disease is thus well correlated with neurotransmitter, enzyme and protein levels. The models successfully mimic several, but not all etiological properties of the disease, allowing us to study the mechanisms of neurodegeneration as well as testing new drugs. The rotenone and 6-OHDA rat and snail in vivo parkinsonian models offer an alternative method for investigation of the molecular mechanisms of neuroprotective agents, including PACAP., Summary: PACAP has a neuroprotective effect in different toxin-induced rat and snail parkinsonian models, acting partially through the same mechanisms.

Understanding the genetic diversity of Aegilops biuncialis, a valuable source of agronomical useful genes, may significantly facilitate the introgression breeding of wheat. The genetic diversity and population structure of 86 Ae. biuncialis genotypes were investigated by 32700 DArT markers with the simultaneous application of three statistical methods— neighbor-joining clustering, Principal Coordinate Analysis, and the Bayesian approach to classification. The collection of Ae. biuncialis accessions was divided into five groups that correlated well with their eco-geographic habitat: A (North Africa), B (mainly from Balkans), C (Kosovo and Near East), D (Turkey, Crimea, and Peloponnese), and E (Azerbaijan and the Levant region). The diversity between the Ae. biuncialis accessions for a phenological trait (heading time), which is of decisive importance in the adaptation of plants to different eco-geographical environments, was studied over 3 years. A comparison of the intraspecific variation in the heading time trait by means of analysis of variance and principal component analysis revealed four phenotypic categories showing association with the genetic structure and geographic distribution, except for minor differences. The detailed exploration of genetic and phenologic divergence provides an insight into the adaptation capacity of Ae. biuncialis, identifying promising genotypes that could be utilized for wheat improvement.

The adverse effects of heat on plant yield strongly depend on its duration and the phenological stage of the crops when the heat occurs. To clarify the effects of these two aspects of heat stress, systematic research was conducted under controlled conditions on 101 wheat cultivars of various geographic origin. Different durations of heat stress (5, 10 and 15 days) were applied starting from three developmental stages (ZD49: booting stage, ZD59: heading, ZD72: 6th day after heading). Various morphological, yield-related traits and physiological parameters were measured to determine the stress response patterns of the wheat genotypes under combinations of the duration and the timing of heat stress. Phenological timing significantly influenced the thousand-kernel weight and reproductive tiller number. The duration of heat stress was the most significant component in determining both seed number and seed weight, as well as the grain yield consequently, explaining 51.6% of its phenotypic variance. Irrespective of the developmental phase, the yield-related traits gradually deteriorated over time, and even a 5-day heat stress was sufficient to cause significant reductions. ZD59 was significantly more sensitive to heat than either ZD49 or ZD72. The photosynthetic activity of the flag leaf was mostly determined by heat stress duration. No significant associations were noted between physiological parameters and heat stress response as measured by grain yield. Significant differences were observed between the wheat genotypes in heat stress responses, which varied greatly with developmental phase. Based on the grain yield across developmental phases and heat stress treatments, eight major response groups of wheat genotypes could be identified, and among them, three clusters were the most heat-tolerant. These cultivars are currently included in crossing schemes, partially for the identification of the genetic determinants of heat stress response and partially for the development of new wheat varieties with better heat tolerance.

Low temperatures are required to regulate the transition from vegetative to reproductive growth via a pathway called vernalization. In wheat, vernalization predominantly involves the cold upregulation of the floral activator VERNALIZATION1 (VRN1). Here, we have used an extreme vernalization response, identified through studying ambient temperature responses, to reveal the complexity of temperature inputs into VRN-A1, with allelic inter-copy variation at a gene expansion of VRN-A1 modulating these effects. We find that the repressors of the reproductive transition, VERNALIZATION2 (VRN2) and ODDSOC2, are re-activated when plants experience high temperatures during and after vernalization. In addition, this re-activation is regulated by photoperiod for VRN2 but was independent of photoperiod for ODDSOC2. We also find this warm temperature interruption affects flowering time and floret number and is stage specific. This research highlights the important balance between floral activators and repressors in coordinating the response of a plant to temperature, and that the absence of warmth is essential for the completion of vernalization. This knowledge can be used to develop agricultural germplasm with more predictable vernalization responses that will be more resilient to variable growth temperatures., Highlighted Article: Analysis of the flowering and growth responses of a range of Triticum aestivum cultivars to warmer growth temperatures reveals an additional layer of temperature regulation in the vernalization response pathway.

In Europe, several exotic Aedes species, such as Aedes albopictus, Aedes eagypti, Aedes japonicas and Aedes koreicus, have become established. Mosquito-borne infection has also become a rising public health issue in Europe. This study aims to present the results of the first Hungarian systematic mosquito monitoring (SMM) operating in an urban environment in the city of P&eacute, cs. It also explains the implementation of a targeted mosquito control by the development of a mosquito map with the SMM approach, thus contributing to a reduction of the risks of mosquito-borne diseases. The mosquito trapping started in 2015, and the traps were Center for Disease Control (CDC) equipment. Based on the data of the SMM, an urban mosquito data set was developed. It consisted of the data of 441 trappings, and the data were statistically analyzed. The results show that three influencing factors impacted mosquito population: distance from running water, built-up density and average temperature had a causal impact on the average number of mosquitoes of an urban area. Each of these factors showed direct impact, and the impact intensified with the combination of the three factors. On that basis, it was made clear which areas of the city mosquito control activity should be focused. Areas of the city where interventions of lower intensity may be necessary compared to the intensity of the earlier mosquito control measures could also be determined. Compared to the previous practice, in which the entire city was involved in intensive mosquito control, now the intensive larviciding is advised to be applied in 42.1% of the total residential areas of the city. Thus, mosquito control of lower intensity could also be sufficient in the remaining 57.9% of the city area. This resource reallocation based on planning can boost the efficiency of the control and lead to a positive change towards sustainability regarding reduced pressure on the environment and reduced expenses of protection.

In this study, we investigate the potentiating effect of PACAP27 on cholinergic neuromuscular transmission in the recently discovered flexor muscles of the land snail, Helix pomatia. Using immunohistochemistry, we show that PACAP and PAC1 receptors are present in nerve fibers innervating the flexor muscles but not in the muscle itself. We also observed that PACAP27 exerts both pre- and postsynaptic effects on the cholinergic synapse and performed tests using a broad spectrum of chemicals in order to explore the possible intracellular pathways through which PACAP mediates its stimulatory effect. Our pharmacological data demonstrate that PACAP27 presynaptically enhances the release of acetylcholine by activating the adenylate cyclase-cAMP-PKA pathway. Postsynaptically, PACAP27 was found to enhance muscle contractility by PKC-mediated signaling pathway resulting in an increased Ca(2+) release from intracellular stores. These findings suggest that regulation of Ca(2+) release may contribute to the stimulatory effect of PACAP. Our data are the first demonstration of the potentiating effect of PACAP27 at the molluscan excitatory neuromuscular contact.

Having been investigated for over 40 years, some aspects of the biology of terrestrial gastropod's olfactory system have been challenging and highly contentious, while others still remain unresolved. For example, a number of terrestrial gastropod species can track the odor of food, while others have no strong preferences toward food odor; rather they find it by random encounter. Here, while assessing the most recent findings and comparing them with earlier studies, the aspects of the food selection based on olfactory cues are examined critically to highlight the speculations and controversies that have arisen. We analyzed and compared the potential role of airborne odors in the feeding behavior of several terrestrial gastropod species. The available results indicate that in the foraging of most of the terrestrial gastropod species odor cues contribute substantially to food finding and selection. The results also suggest, however, that what they will actually consume largely depends on where they live and the species of gastropod that they are. Due to the voluminous literature relevant to this object, this review is not intended to be exhaustive. Instead, I selected what I consider to be the most important or critical in studies regarding the role of the olfaction in feeding of terrestrial gastropods.

The presence of a mixture of progestogens at ng/L concentration levels in surface waters is a worldwide problem. Only a few studies explore the effect of progestogen treatment in a mixture as opposed to individual chemicals to shed light on how non-target species respond to these contaminants. In the present study, we used an invertebrate model species, Lymnaea stagnalis, exposed to a mixture of four progestogens (progesterone, levonorgestrel, drospirenone, and gestodene) in 10ng/L concentration for 3 weeks. Data at both physiological and cellular/molecular level were analyzed using the ELISA technique, stereomicroscopy combined with time lapse software, and capillary microsampling combined with mass spectrometry. The treatment of adult Lymnaeas caused reduced egg production, and low quality egg mass on the first week, compared to the control. Starting from the second week, the egg production, and the quality of egg mass were similar in both groups. At the end of the third week, the egg production and the vitellogenin-like protein content of the hepatopancreas were significantly elevated in the treated group. At the cellular level, accelerated cell proliferation was observed during early embryogenesis in the treated group. The investigation of metabolomic changes resulted significantly elevated hexose utilization in the single-cell zygote cytoplasm, and elevated adenylate energy charge in the egg albumen. These changes suggested that treated snails provided more hexose in the eggs in order to improve offspring viability. Our study contributes to the knowledge of physiological effect of equi-concentration progestogen mixture at environmentally relevant dose on non-target aquatic species.

A complete SGP null mutant (Sgp-A1B1D1 null) winter wheat line was crossed with three wheat varieties: ‘Ukrainka’, ‘Lona’ and ‘Solstice’. Three backcrosses (BCs) were carried out and marker assisted selection used to identify lines carrying all the three mutant alleles for each cross. BC3 and BC2xBC2 F3, F4 and F5 lines (2012-2014) were studied in detail for physical (thousand kernel weight), compositional (protein, gluten, amylose) and starch viscosity properties (rapid visco analyser-RVA). Ten triple mutant high-amylose lines (∼40%) were identified among the BC2xBC2 lines, which had high protein content (>14%), low starch content, gluten index, starch viscosity values and thousand kernel weight. Two of these lines had moderately high amylose (∼30%) content, with outstanding gluten quality, but modified starch viscosity properties.

The precise elimination of selectable marker genes is highly desirable, when their function is no longer needed, because their presence raised worldwide public concerns against the release of genetically modified plants. This is the first report of simultaneous application of the minimal gene cassette and cold-inducible Cre/lox recombination system in wheat. The bar selection and cre-recombinase genes were eliminated from T0 and T1 transgenic lines with 44 and 51 % efficiency. This approach provides a new, reasonably effective technique to produce selection gene-free transgenic wheat lines either immediately after tissue culture, or from the subsequent transgenic generation. The advantage of this method is that it does not require any additional cold treatment during the plant regeneration/growing because the transgene elimination is ensured by the vernalisation. Application of this method prevents gene flow by pollen and seed, because the selection and recombinase genes are eliminated before pollen development, therefore reducing the risk of GM plants.

In the present study, heat treatment was carried out in five different phenological phases, from the first node detectable (DEV31) growth stage to 20 days after flowering, on four wheat genotypes with very different adaptation strategies. They were grown in a controlled environment in a phytotron chamber and exposed to a night temperature of 20°C and a day temperature of either 30°C, at DEV31, or 35°C at all the later developmental phases, for an interval of 14 days. Plant height, leaf number, number of tillers, grain number and grain weight per main and side spikes, TKW per main and side spikes, length of the main and side spikes, and spikelet number per main and side spikes were recorded. High temperature enhanced the stem growth intensity, plant height and tiller number. In contrast, the length of side spikes, spikelet no./side spike, grain no./main and side spike, grain weight/main and side spike and TKW/main and side spike were significantly decreased. The stress response depended strongly on the developmental phase in which the heat stress was applied. Fleischmann 481 and Soissons showed definitely contrasting tendencies both in grain number and grain weight. In the case of the Plainsman V and Mv Magma pair, the higher heat stress tolerance of Magma compared to Plainsman V was evident also from the grain number and weight of the main spike at each developmental phase.

Studies on plant development phases and yield component patterns of wheat are essential for a better understanding of adaptation in wheat. Our main aim was to carry out detailed phenological analyses of 18 wheat genotypes in three sowing times for determining the effect of sowing date on individual phenophases, and yield components. Sowing date had the single greatest effect on the start of intensive stem elongation. The longer vegetation period had a favourable effect on main spike length and on the spikelet number per spike, but had no influence on thousand-kernel weight and grain number per spike. The time between the first node appearance and start of intensive stem elongation had a significant effect on the number of reproductive tillers. A close association (R2 = 0.191) was observed during the second phase of intensive stem elongation between the boot stage-to-heading interval and the number of spikelets per spike. Two-way analysis of variance on the yield components showed that the sowing date, as a main factor, had a weaker effect on the phenophases than on morphological and developmental parameters. The insensitive allele of the Ppd-D1 gene shortened the time required for first node appearance and heading both in autumn and spring sowing.

The distribution and functional presence of three voltage-dependent potassium channels, K v 2.1, K v 3.4, K v 4.3, respectively, were studied in the central nervous system of the snail Helix pomatia by immunohistochemical and electrophysiological methods. Cell clusters displaying immunoreactivity for the different channels were observed in all parts of the CNS, although their localization and number partly varied. Differences were also found in their intracellular, perikaryonal and axonal localization, as well as in their presence in non-neuronal tissues nearby the CNS, such as the perineurium and the aorta wall. At ultrastructural level K v 4.3 channel immunolabeling was observed in axon profiles containing large 80–100 nm granular vesicles. Blotting analyses revealed specific signals for the K v 2.1, K v 3.4 and K v 4.3 channels, confirming the presence of the channels in the Helix CNS. Voltage-clamp recordings proved that outward currents obtained from neurons displaying K v 3.4 or K v 4.3 immunoreactivity contained transient components while K v 2.1 immunoreactive cells were characterized by delayed currents. The distribution of the K + -channels containing neurons suggests specific roles in intercellular signaling processes in the Helix CNS, most probably related to well-defined, partly local events. The cellular localization of the K + -channels studied supports their involvement in both pre- and postsynaptic events at perikaryonal and axonal levels.

Heading of cereals is determined by complex genetic and environmental factors in which genes responsible for vernalization and photoperiod sensitivity play a decisive role. Our aim was to use diagnostic molecular markers to determine the main allele types in VRN-A1, VRN-B1, VRN-D1, PPD-B1 and PPD-D1 in a worldwide wheat collection of 683 genotypes and to investigate the effect of these alleles on heading in the field. The dominant VRN-A1, VRN-B1 and VRN-D1 alleles were present at a low frequency. The PPD-D1a photoperiod-insensitive allele was carried by 57 % of the cultivars and was most frequent in Asian and European cultivars. The PPD-B1 photoperiod-insensitive allele was carried by 22 % of the genotypes from Asia, America and Europe. Nine versions of the PPD-B1-insensitive allele were identified based on gene copy number and intercopy structure. The allele compositions in PPD-D1, PPD-B1 and VRN-D1 significantly influenced heading and together explained 37.5 % of the phenotypic variance. The role of gene model increased to 39.1 % when PPD-B1 intercopy structure was taken into account instead of overall PPD-B1 type (sensitive vs. insensitive). As a single component, PPD-D1 had the most important role (28.0 % of the phenotypic variance), followed by PPD-B1 (12.3 % for PPD-B1_overall, and 15.1 % for PPD-B1_intercopy) and VRN-D1 (2.2 %). Significant gene interactions were identified between the marker alleles within PPD-B1 and between VRN-D1 and the two PPD1 genes. The earliest heading genotypes were those with the photoperiod-insensitive allele in PPD-D1 and PPD-B1, and with the spring allele for VRN-D1 and the winter alleles for VRN-A1 and VRN-B1. This combination could only be detected in genotypes from Southern Europe and Asia. Late-heading genotypes had the sensitivity alleles for both PPD1 genes, regardless of the allelic composition of the VRN1 genes. There was a 10-day difference in heading between the earliest and latest groups under field conditions. Electronic supplementary material The online version of this article (doi:10.1007/s11032-014-0034-2) contains supplementary material, which is available to authorized users.

The heading characters and morphological traits of two partial sets of wheat–barley disomic addition lines, namely Mv9kr1/Igri and Asakaze/Manas, were evaluated under controlled environmental conditions in a phytotron under long-day, short-day and non-vernalised conditions and in field-sown experiments. The winter barley chromosome additions significantly influenced the flowering time of wheat both in the controlled environment test and under field-sown conditions. Of all the barley addition lines, the effect of the 4H and 7H additions was the most characteristic. The 7H addition lines were the earliest in both cultivar combinations in each treatment. In the Mv9kr1/Igri combination the 4H addition was the latest under all the environmental conditions. In the Asakaze/Manas combination 4H addition was the latest under short-day and long-day illumination in the phytotron but the 6H addition was the latest without vernalisation and in the field in 2012. There was 12 and 11 days difference between the flowering times of the 7H and 4H Mv9kr1/Igri and Asakaze/Manas addition lines in the field in 2012, which increased to 52 and 44 days under short-day illumination in the phytotron. In the winter wheat background, the addition of 2H carrying the photoperiod sensitivity gene Ppd-H1 decreased the flowering time under the short photoperiod regime, but had a very strong delaying effect under field-grown conditions. Considering the yield components under field conditions, 4H was the most fertile of the addition lines, while 7H showed the highest tillering capacity, and Igri 3H had good tillering capacity and the highest number of seeds per plant.

Ambient temperature plays an important role in plant development. In cereals, little is known about the exact effects of ambient temperature in the range between it being a vernalising agent and an abiotic stress factor; thus the genetic determinants involved in the registering and response to ambient temperature, and their natural variation has not been dissected either. Principally, we wished to establish the level of natural variation in response to ambient temperature in barley via studying plant phenological development. The responses to temperature of 168 barley genotypes of different provenances and seasonal growth habit groups were observed in controlled environments. The effects of four temperature regimes (13 ◦ C, 16.5 ◦ C, 18 ◦ Ca nd 23 ◦ C) on the duration of plant phenophases were examined. The plant development was characterised in a series of consecutive phenophases that span the plant life cycle from germination through flowering to attainment of maximum plant height. Ambient temperature affected significantly plant development, with substantial variation in responses among the genotypes. Six major types of responses were identified, which depended strongly on seasonal growth habit, with only a small degree of overlap. Although the differences in the timing of development among clusters were significant under each temperature regime, the 23 ◦ C treatment resulted in the largest diversity of responses, with significant changes in the ranking of the six clusters compared to other treatments. Two clusters showed particularly unusual responses to 23C: the development of one winter barley cluster was extremely accelerated by the 23C treatment, whereas the development of one spring barley cluster was significantly delayed. Ambient temperature assumes importance as a regulatory cue in the intricate and complex temporal and spatial regulation network of plant development in cereals and acts mostly through its regulatory effect on certain developmental phases such as the onset and duration of the intensive stem elongation.

Stress tolerance is associated with the activation of antioxidant compounds and enzyme systems that are capable of neutralising the reactive oxygen species (ROS) continually produced in response to stress. The present experiment was designed to compare the heat tolerance of four winter wheat varieties in the shooting and grain-filling stages by investigating changes detected in antioxidant enzyme activity and yield components in response to heat stress.Heat treatment was found to cause a significant rise in the activity of the glutathione-S-transferase and catalase enzymes, while there was usually a less intense decline in the activity of guaiacol peroxidase.An analysis of yield data revealed that heat stress had a more pronounced effect during grain filling in this experiment than at the beginning of shooting, as shown by the greater reduction in thousand-kernel weight and yield.

Changing climatic conditions with warming winters and shifts in the frequencies of drought, intense rainfall and cold spells together with associated changes in the geographical distribution of arable crops increase the challenges for selecting new varieties. In this context, we aim to contribute to a better understanding of the determinants of barley (Hordeum vulgare) frost tolerance (FRT) and consequent improvements to marker-assisted selection (MAS). Freezing injury in a diversity panel of 121 barley genotypes with different growth habits and origins was assessed using phenotyping based on chlorophyll fluorescence (Fv/Fm) measurements to screen genetic diversity in plants at an early growth stage. The haplotypes of vernalisation and photoperiod genes were determined with PCR, and correlation analyses were done using data from 12 laboratory and field-laboratory FRT tests. Previous results of allelic combinations of VRN-H1/VRN-H2 for FRT were confirmed with these experiments using a larger set of genotypes. The predictive power of polymorphisms in VRN-H1 intron 1 region for FRT was significantly higher than that of the VRN-H1 promoter polymorphism. The vrn-H1/vrn-H2 facultative genotypes had similar or higher FRT than vrn-H1/Vrn-H2 winter genotypes under suboptimal hardening conditions. Genes regulating long-day and short-day photoperiodic responses were significantly correlated with FRT. The most parsimonious model for prediction of FRT was based on polymorphisms in the VRN-H1 intron 1 region, VRN-H2 and PPD-H2 and explained 69% of the variation in FRT.

In our previous study, we measured 0.23-13.67ng/L progestogens (progesterone, drospirenone, levonorgestrel) in natural waters in the catchment area of the largest shallow lake of Central Europe, Lake Balaton. Progestogen contaminations act as potent steroids with mixed progestagenic, androgenic and mild estrogenic effects that is why our aim was to investigate the morphological and molecular effects of mixture of progesterone, drospirenone, and levonorgestrel in environmentally relevant (10ng/L) and higher (50 and 500ng/L) exposure concentrations in common roach, Rutilus rutilus. Steroids (e.g. progestogens) and the protein deglycase DJ-1 chaperon molecule aim the same target molecules in cells, therefore, we hypothesized that a relationship may exist between progestogens and DJ-1. Furthermore, our other aim was to follow the changes of signal molecules of different biological function due to progestogen treatment in serum and brain. Adult roaches were exposed to 10, 50 and 500ng/L of mixture of progestogen for 42 days and their somatic indices (brain-somatic, liver-somatic, gonadosomatic and kidney-somatic) were measured. Vitellogenin (VTG) expression (estrogen effect) or inhibition (androgen effect) in fish is a widely used biomarker so we measured its changes in liver by ELISA. To determine the quantity and to map the spatial distribution of DJ-1 chaperon protein the brain and liver tissues were analyzed by ELISA and immunohistochemistry. Furthermore, we also studied molecular alterations: a) in the serum by measuring cholesterol, low-density lipoprotein (LDL), high-density lipoprotein (HDL) and triglyceride concentrations and b) in brain homogenate using a cell stress array kit (26 protein). The somatic index of liver and kidney significantly in all the treated groups, whereas the gonadosomatic index of 500ng/L treated group showed significant decrease compared to control animals. VTG level increased significantly in 500ng/L progestogen treated group. Since the concentration of DJ-1 significantly increased in brain and liver in all progestogen treatment groups, the DJ-1 protein could be able to a more sensitive marker than VTG. Serum LDL and cholesterol levels of exposed fish were significantly decreased. DJ-1 was mediated through the stimulation of the expression of LDL-receptor which facilitates reuptake subsequently. In summary, our observations unfolded new data about molecular alterations induced by the combined action of environmental progestogens. In addition, the DJ-1 chaperon protein as a possible biomarker helped to trace the abiotic chemical environmental contaminations, like progestogens in the freshwater ecosystems.

The occurrence and distribution of pituitary adenylate cyclase-activating polypeptide (PACAP) are summarized in invertebrate species with special attention to annelids, mollusks, and arthropods in this review. Furthermore, the role of PACAP is highlighted in physiological and behavioral processes of oligochaete (Lumbricus), gastropods (Helix, Lymnaea), insect (Drosophila), as well as malacostraca (Litopenaeus). Since its discovery PACAP has become increasingly recognized for its important and diversified roles in the central and peripheral nervous system and in several peripheral organs of a variety of vertebrate and invertebrate species. Twenty-six years after its discovery, PACAP is now one of the most extensively studied neuropeptides both in invertebrate and vertebrate species. This review surveys the importance of PACAP or PACAP-like peptide(s) in invertebrates. The relevance of studies on lower vertebrates and invertebrates, which do not have a pituitary gland like higher vertebrate, is to contribute to the unraveling of fundamental effects of PACAP or PACAP-like peptide(s) and to provide a comparative view.

Breeding of new winter wheat cultivars with good heat tolerance requires better understanding of the genetic background of heat tolerance. In the present work the effect of heat stress on the 6th day after heading was investigated in a doubled haploid (DH) population arising from a cross between heat-sensitive (Plainsman V) and heat-tolerant (Mv Magma) cultivars. Averaged over the population, heat stress was found to result in a significant reduction in biomass, grain yield and grain number per plant, and in thousand-kernel weight (TKW) and harvest index. High temperature had the greatest effect on the grain yield, with a drop of 36.2% compared with the control. This could be attributed jointly to significant reductions in the TKW of the main ear and in the grain number of the side tillers. The relationship between the yield parameters was confirmed by the positive correlations obtained for the lines in the population. The diverse levels of heat tolerance in the different lines were confirmed by the significant differences in the reduction in the chlorophyll content (SPAD index) of the flag-leaves and in yield parameters. The changes in yield components in stress condition, however, can be still the most effective tools for heat stress evaluation.

The transitions between various developmental phases are critical in determining the ecological adaptation and yield of cereals. In order to elaborate a methodology for establishing the timing of the consecutive plant developmental phases from germination to the fully developed plant, regular measurements of changes in developmental components were carried out on one winter (Kompolti Korai) and one spring (Morex) barley cultivar in a model experiment. Under the controlled environmental conditions linear regression was characteristic of the associations between the chronological time and all or most of the time course data of plant height, tiller and leaf numbers. The initial growth of the spring barley was twice as intensive as that of the winter barley. The length of the stem elongation phases was similar for the two varieties, but the winter barley cultivar showed significantly more intensive stem growth compared to the spring barley. The spring barley reached all the plant developmental phases significantly earlier than the winter barley. For both cultivars, tillering continued till after first node appearance and there was a definite delay between first node appearance and the beginning of the stem elongation phase. The determination of the full series of phenophases, together with the evaluation of various yield components on the same plant, provide an excellent way of establishing plant developmental patterns and may make a significant contribution to achieving a better understanding of the associations between plant developmental patterns and the adaptation and yielding ability of cereals.

Among the factors which determine yield reliability an important role is played by disease resistance. One of the breeding aims in the Martonvásár institute is to develop wheat varieties with resistance to major diseases. The winter wheat varieties bred in Martonvásár are examined in artificially inoculated nurseries and greenhouses for resistance to economically important pathogens. The effectiveness of designated genes for resistance to powdery mildew and leaf rust has been monitored over a period of several decades. None of the designated major resistance genes examined in greenhouse tests is able to provide complete resistance to powdery mildew; however, a number of leaf rust resistance genes provide full protection against pathogen attack (Lr9, Lr19, Lr24, Lr25, Lr28 and Lr35). In the course of marker-assisted selection, efficient resistance genes (Lr9, Lr24, Lr25 and Lr29) have been incorporated into Martonvásár wheat varieties. The presence of Lr1, Lr10, Lr26, Lr34 and Lr37 in the Martonvásár gene pool was identified using molecular markers. New sources carrying alien genetic material have been tested for powdery mildew and leaf rust resistance. Valuable Fusarium head blight resistance sources have been identified in populations of old Hungarian wheat varieties. Species causing leaf spots (Pyrenophora tritici-repentis, Septoria tritici and Stagonospora nodorum) have gradually become more frequent over the last two decades. Tests on the resistance of the host plant were begun in Martonvásár four years ago and regular greenhouse tests on seedlings have also been initiated.

Programmed cell death leading to apoptosis is essential for normal development and homeostasis in plants and throughout the animal kingdom. Although there are differences in apoptotic mechanisms between lower animals and vertebrates, crucial biochemical components of the programmed cell death pathways remained remarkably conserved throughout evolution. Despite decades of studies on the neurobiology and development of mollusks, comparatively little is known about the mechanisms of apoptosis in this phylum. In this review, an attempt is made to summarize data obtained on mollusks so far, and to discuss the molecular mechanisms, the functional and ecological significance of apoptosis and the advantages of snail preparations as tools for programmed cell death research. A definitive comparison of the data obtained on mollusks with those obtained on the more widely studied vertebrates, will contribute to the better understanding of the apoptotic process in general and of its evolutionary development.

Background information. PCD (programmed cell death) is a common mechanism to remove unwanted and excessive cells from organisms. In several exocrine cell types, PCD mode of release of secretory products has been reported. The molecular mechanism of the release, however, is largely unknown. Our aim was to study the molecular mechanism of saliva release from cystic cells, the specific cell type of snail SGs (salivary glands). Results. SG cells in active feeding animals revealed multiple morphological changes characteristic of PCD. Nerve stimulation and DA (dopamine) increased the number of TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling)-positive cells both in inactive and feeding animals. The DA-induced PCD was prevented by TEA (tetraethylammonium chloride) and eticlopride, emphasizing the role of K channels and D2 receptors in the PCD of cystic cells. DA enhanced cyto-c (cytochrome c) translocation into the cytosol and methyl-β-cyclodextrin prevented it, suggesting apoptosome formation and ceramide involvement in the PCD linking of the surface DA receptor to mitochondria. Western blot analysis revealed that the release of cyto-c was under the control of Bcl-2 and Bad. DA also increased the active caspase-3 in gland cells while D2 receptor antagonists and TEA attenuated it. Conclusion. Our results provide evidence for a type of transmitter-mediated pathway that regulates the PCD of secretory cells in a mitochondrial-caspase-dependent manner. The activation of specific molecules, such as K channels, DA receptors, cyto-c, ceramide, Bcl-2 proteins and caspase-3, but not caspase-8, was demonstrated in cells involved in the DA-induced PCD, suggesting that PCD is a physiological method for the release of saliva from SG cells.

Pituitary adenylate cyclase activating polypeptide (PACAP) shows a remarkable sequence similarity among species and several studies provide evidence that the functions of PACAP have also been conserved among vertebrate species. Relatively little is known about its presence and functions in invertebrates. The aim of the present study was to investigate whether the well-known anti-apoptotic effect of PACAP can also be demonstrated in invertebrates. This effect was studied in the salivary gland of a molluscan species, Helix pomatia. In this work, we first showed the presence of PACAP-like immunoreactivity in the Helix salivary gland by means of immunohistochemistry. Radioimmunoassay measurements showed that PACAP38-like immunoreactivity dominated in the salivary gland of both active and inactive snails and its concentration was higher in active than in inactive animals in contrast to PACAP27-like immunoreactivity, which did not show activity-dependent changes. PACAP induced a significant elevation of cAMP level in salivary gland extracts. Application of apoptosis-inducing agents, dopamine and colchicine, led to a marked increase in the number of terminal uridine deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive apoptotic cells in the salivary gland, which was significantly attenuated by PACAP treatment. In a similar manner, the number of caspase-positive cells was reduced after co-application of dopamine and PACAP. Taken together, the data indicate that PACAP activates cAMP in a molluscan species and we show, for the first time, that PACAP is anti-apoptotic in the invertebrate Helix pomatia.

GPRCs are regulated via phosphorylation by different protein kinases including GRKs and PKA and PKC. The purpose of this study was to determine the presence and physiological role of GRKs in the tissues of the snail, Helix pomatia . Here we report that immunoblotting of brain homogenate with anti-GRK2/3 antibody prepared from mammalian tissue can be detected in snail GRK-like immunoreactivity. The GRK2/3 immunoreactivity was found at ∼ 80 kDa in a variety of cells, including salivary duct, salivary gland and eye. Intracellular injection of the anti-GRK2/3 prevented the neuron from desensitization and agonist-induced activation augmented the phosphorylated GRKs in the membrane fraction suggesting that GRKs may have a functional role in the neuropeptide receptor desensitization in snail.

The aim of the present study was to assess the cellular mechanism of secretion in the salivary gland of the snail, Helix pomatia, using electrophysiological, electron microscopic and immunohistochemical techniques. A homogeneously distributed membrane potential (-56.6 +/- 9.8 mV) was determined mainly by a K+ -electrochemical gradient and partly by the contribution of the electrogenic Na+ -pump and Cl- conductance. Low resistance electrical coupling sites were identified physiologically. Transmission electron microscopy and innexin 2 antibody revealed the presence of gap-junction-like membrane structures between gland cells. It is suggested that gap-junctions are sites of electrotonic intercellular communication, which integrate the gland cells into a synchronized functional unit in the acinus. Stimulation of the salivary nerve elicited secretory potentials (depolarization) which could be mimicked by local application of acetylcholine, dopamine or serotonin. In voltage-clamp experiments four major conductances were identified: a delayed rectifier (IK), a transient (IA) and a Ca2+ -activated outward K+ current (IK(Ca)) and Ca2+ -inward currents (ICa). It is suggested that one or more of these conductances may give rise to a stimulus activated secretory potential leading to excitation-secretion coupling and subsequent the release of the mucus from the gland cells.

Members of the mytilus inhibitory peptide (MIP) family play an important role in the modulation of many physiological processes in molluscs. The signal transduction pathways affected by the MIP effect have not, however, been elucidated. Application of guanosine 5'-[gamma-thio]triphosphate tetralithium salt (GTPgammaS), guanosine 5'-[beta-thio]diphosphate trilithium salt (GDPbetaS), the G-protein inhibitor suramin and pertussis toxin (PTX) demonstrated the involvement of the PTX-insensitive G-protein in the signal transduction pathway mediating MIP effects. Both G-protein alpha(i) and betagamma subunits were identified in D-neurons of Helix pomatia by immunoblotting. Their role in signal transduction was shown in electrophysiological experiments, which supported the notion that, in addition to the Galpha subunit, the betagamma dimer also participates in the neuropeptide-induced activation of K-channels in snail neurons. Finally, neuropeptide-activated responses were inhibited by the activation of adenylyl cyclase and by blockers of the phospholipase pathway. We suggest that bifurcation of the signal transduction takes place at the level of G-protein subunits. The alpha subunit may have a direct effect on adenylyl cyclase, while the betagamma subunit may have a direct effect on phospholipase enzymes.

Blocking of the GABA-activated chloride current by cadmium was investigated in identified nerve cells (RPeD1, RPaD1) of the pond snail (Lymnaea stagnalis L.), using a two-microelectrode voltage-clamp technique. Cd2+, at 50 μM extracellular concentration, inhibited GABA-activated chloride currents, both in normal and Ca2+-free solution. Intracellular injection of Ca2+ or the application of caffeine mimicked the inhibitory effect of Cd2+ on GABA-elicited currents. Cd2+-block was eliminated, or it was substantially decreased, when neurons were intracellularly loaded with EGTA, or when the Ca2+-release was blocked by ruthenium red. The blocking effect of Cd2+ was also eliminated by applying G-protein inhibitors: pertussis toxin, suramin or GTP-γ-S. Finally, intracellularly injected Cd2+ was ineffective at eliciting an inward current on GABA-activated currents, suggesting that the Cd2+-binding site resides extracellularly. These results suggest that cadmium inhibited GABA-activated chloride currents by increasing the intracellular Ca2+ level, by releasing it from intracellular stores and by interacting with a putative G-protein-coupled cell-surface “metal-receptor”.

Distribution, chemical-neuroanatomy, concentration, and uptake-release properties of histamine (HA)-containing neurons and the possible physiological effects of HA in the central and peripheral nervous system of the pulmonate snails, Helix pomatia and Lymnaea stagnalis, are described. In the CNS of both species, the distribution pattern of HA-immunoreactive (HA-IR) neurons was similar. In both species the majority were located in the buccal, cerebral, and pedal ganglia. In Helix, approximately 400 HA-IR neurons were seen, whereas in Lymnaea approximately 130 labeled cells were visualized. The neuropils, connectives, commissures, several peripheral nerves, and a part of the peripheral tissues (lip and foot of both species and the upper tentacles of Helix) were innervated by HA-IR elements. Numerous sensory cells were found in the tentacles, lip, and statocysts. The HA concentration values assayed by HPLC ranged from 4.8 to 47.4 pmol/mg in the different central ganglia of Helix, and from 4.3 to 18.6 pmol/mg in Lymnaea CNS, whereas the peripheral tissues contained 0.33-1 pmol/mg HA in Helix and 0.26-0.46 pmol/mg in Lymnaea. In the Lymnaea CNS, a high-affinity (37.6 microM), single component 3H-HA uptake system was demonstrated. 3H-HA release evoked by either electrical stimulation or 100 mM K+ could be prevented in Ca2+-free physiological solution. Voltage-clamp experiments indicated specific changes caused by HA in the membrane conductance of identified central neurons of Helix and Lymnaea. Exogenously applied 10(-5) M HA resulted in the acceleration of locomotion (gliding by foot cilia) of Lymnaea. The findings suggest an important signaling role of HA, described here for the first time, in the nervous system of higher-order, pulmonate, gastropods, involving efferent, integrative, and sensory functions. The data can also be applied as a background for further specification of HA in the regulation of different behaviors in these species.

The block of the transient outward K-current, I(K(A)) by 4-aminopyridine (4-AP) and blood-depressing substances (BDS) was investigated in identified Helix pomatia neurons (LPa3) using the two microelectrode voltage-clamp technique. The present study shows that 4-AP inhibits I(K(A)) in snail neurons in a voltage- and concentration-dependent manner. The 4-AP block of I(K(A)) involves the block of both open and closed states of the channel, however binding to open channels is preferred. It is suggested that 4-AP have two binding sites on the identified Helix neuron. One site causes an open channel block, which affects the N-type inactivation, and binding to the second site induces closed channel block, which affects C-type inactivation. In control solution the inactivating phase of the current is biexponential, suggesting simultaneous presence of two types of inactivation. The counterplay of these mechanisms results in the crossover of the current traces recorded from control and 4-AP blocked channels. It is assumed that use-dependence does not occur through blocker 'trapping', but rather by a different mechanism. BDS had no effect on Helix I(K(A)), suggesting that transient potassium channels in LPa3 neuron are not Kv3.4 type channels.

Three flexor muscles of the posterior tentacles of the snail Helix pomatia have recently been described. Here, we identify their local motor neurons by following the retrograde transport of neurobiotin injected into these muscles. The mostly unipolar motor neurons (15–35 µm) are confined to the tentacle digits and send motor axons to the M2 and M3 muscles. Electron microscopy revealed small dark neurons (5–7 µm diameter) and light neurons with 12–18 (T1 type) and 18–30 µm diameters (T2 type) in the digits. The diameters of the neurobiotin-labeled neurons corresponded to the T1 type light neurons. The neuronal processes of T1 type motor neurons arborize extensively in the neuropil area of the digits and receive synaptic inputs from local neuronal elements involved in peripheral olfactory information processing. These findings support the existence of a peripheral stimulus–response pathway, consisting of olfactory stimulus—local motor neuron—motor response components, to generate local lateral movements of the tentacle tip (“quiver”). In addition, physiological results showed that each flexor muscle receives distinct central motor commands via different peritentacular nerves and common central motor commands via tentacle digits, respectively. The distal axonal segments of the common pathway can receive inputs from local interneurons in the digits modulating the motor axon activity peripherally without soma excitation. These elements constitute a local microcircuit consisting of olfactory stimulus—distal segments of central motor axons—motor response components, to induce patterned contraction movements of the tentacle. The two local microcircuits described above provide a comprehensive neuroanatomical basis of tentacle movements without the involvement of the CNS.

Hibernation, as behavior, is an evolutionary mode of adaptation of animal species to unfavorable environmental conditions. It is generally characterized by suppressed metabolism, which also includes down regulation of the energy consuming ion-channel functioning. Experimental data regarding decreased ion-channel function are scarce. Therefore, our goal was to study the possible down regulation of voltage-gated sodium channel (NaV) subtypes in the neurons of hibernating snails. Our immunohistochemical experiments revealed that the expression of NaV1.8-like channels in the central nervous system was substantially down regulated in hibernating animals. In contrast to NaV1.8-like, the NaV1.9-like channels were present in neurons independently from hibernating and non-hibernating states. Our western blot data supported the immunohistochemical results according to which the band of the NaV1.8-like channel protein was less intensively labeled in the homogenate of the hibernating snails. The NaV1.9-like immunoreactivity was equally present both in hibernating and active snails. Micro-electrophysiological experiments show that in hibernating snails both NaV1.8- and NaV1.9-like currents are substantially decreased compared to that of the active snails. The contradictory electrophysiological and immunohistochemical or western blot data suggest that the molecular mechanisms of the "channel arrest" could be different in diverse NaV channel subtypes. Climate changes will affect temperature extremes and a question is how different species beyond their physiological tolerance will or able to adapt to changing environment. Hibernation is an important mode of adaptation to extreme climatic variations, and pursuant to this the present results may contribute to the study of the behavioral ecology.

Effects of seven, pressure applied MIP (Mytilus inhibitory peptides) had been studied on D-neurons of the CNS of Helix pomatia in voltage-clamp experiments. In physiological saline, the peptides produced a hyperpolarization usually coupled with the cessation of any spontaneous spiking activity. Clamped at the resting potential ( approximately -60 mV), peptide applications elicited an outward current, which increased its amplitude by shifting the holding potential towards depolarisation. The response was concentration-dependent and accompanied by an increased membrane conductance. Reversal potentials obtained at different [K+]o were plotted with a slope of 52 mV per ten-fold change in [K+]o showing that the peptide-elicited current was mainly due to the increased K+-conductance(s). The peptide-induced outward current could partially be blocked by Ba2+ (5 mM), CdCl2 (1 mM), TEACl (10 mM) or apamin (2.5x10(-5) M) or furosemide (10 mg/ml) and decreased either in Na+-free or Cl--free solutions. 4-Aminopyridine at 5 mM concentration completely blocked the peptide-induced current. In the presence of high [K+]o, the peptide(s) was still found to induce an outward current at membrane potentials beyond K+-reversal potential. This component was not present in Cl--free saline, suggesting that the current was due to the inward flow of Cl- ions. Our results show that the MIPs have at least two (three) independent actions, each associated with different voltage-, concentration-dependence and ionic mechanisms. It is suggested, that the peptide-induced currents are carried by K+, and Cl- ions. According to our present finding, the observed effects are mediated by the same receptor, activating different second messenger systems, inducing multiple conductance changes in the membrane of neurons of the snail ganglia.

Effects of veratrum alkaloids were studied on the Na-channels of the land snail Helix pomatia. It was found that veratridine and its analogues depolarize the membrane due to the increased Na-permeability. The inactivation was shifted right along the voltage axis and the recovery from the inactivation was faster after veratridine treatment. After alkaloid treatment the selectivity of the Na-channel decreased, however, the selectivity sequence was not altered. The activation curve was not shifted. Veratridine derivatives, which appeared to be more effective on insects, had almost no effect on the Na-current.

Recently, three novel flexor muscles (M1, M2 and M3) in the posterior tentacles of the snail have been described, which are responsible for the patterned movements of the tentacles of the snail, Helix pomatia. In this study, we have demonstrated that the muscles received a complex innervation pattern via the peritentacular and olfactory nerves originating from different clusters of motoneurons of the cerebral ganglia. The innervating axons displayed a number of varicosities and established neuromuscular contacts of different ultrastructural forms. Contractions evoked by nerve stimulation could be mimicked by external acetylcholine (ACh) and glutamate (Glu), suggesting that ACh and Glu are excitatory transmitters at the neuromuscular contacts. Choline acetyltransferase and vesicular glutamate transporter immunolabeled axons innervating flexor muscles were demonstrated by immunohistochemistry and in Western blot experiments. Nerve- and transmitter-evoked contractions were similarly attenuated by cholinergic and glutamatergic antagonists supporting the dual excitatory innervation. Dopamine (DA, 10−5 M) oppositely modulated thin (M1/M2) and thick (M3) muscle responses evoked by stimulation of the olfactory nerve, decreasing the contractions of the M1/M2 and increasing those of M3. In both cases, the modulation site was presynaptic. Serotonin (5-HT) at high concentration (10−5 M) increased the amplitude of both the nerve- and the ACh-evoked contractions in all muscles. The relaxation rate was facilitated suggesting pre- and postsynaptic site of action. Our data provided evidence for a DAergic and 5-HTergic modulation of cholinergic nerves innervating flexor muscles of the tentacles as well as the muscles itself. These effects of DA and 5-HT may contribute to the regulation of sophisticated movements of tentacle muscles lacking inhibitory innervation.

The procerebrum (PC) of the snail brain is a critical region for odor discrimination and odor learning. The morphological organization and physiological function of the PC has been intensively investigated in several gastropod species; however, the presence and distribution of ion channels in bursting and non-bursting cells has not yet been described. Therefore, the aim of our study was to identify the different ion channels present in PC neurons. Based on whole cell patch-clamp and immunohistochemical experiments, we show that Na+-, Ca2+-, and K+-dependent voltage-gated channels are differentially localized and expressed in the cells of the PC. Different Na-channel subtypes are present in large (10–15 μm) and small (5–8 μm) diameter neurons, which are thought to correspond to the bursting and non-bursting cells, respectively. Here, we show that the bursting neurons possess fast sodium current (I NaT) and NaV1.9-like channels and the non-bursting neurons possess slow sodium current (I NaT) and NaV1.8-like channels in addition to the L-type Ca−, KV4.3 (A-type K-channel) and KV2.1 channels. We suggest that the bursting and/or non-bursting character of the PC neurons is at least partly determined by the battery of ion-channels present and their cellular and subcellular compartmentalization.

The purpose of this review is to highlight the role of pituitary adenylate cyclase-activating polypeptide (PACAP) in a range of physiological and behavioral processes of gastropod molluscs, Helix and Lymnaea. Since its discovery in 1989 PACAP has become increasingly recognized for its important and diversified roles in the central and peripheral nervous system and in several peripheral organs of a variety of vertebrate and invertebrate species. Twenty-two years after its discovery, PACAP is now one of the most extensively studied of the neuropeptides. This review surveys the importance of PACAP and PACAP-like peptides in invertebrates, focusing mainly on the gastropod molluscs. The relevance of studies on lower vertebrates and invertebrates, which do not have a pituitary gland, is to contribute to the unraveling of fundamental effects of PACAP or PACAP-like peptides and to provide a comparative view.

Neuropeptides, the most diverse group of signaling molecules, are responsible for regulating a variety of cellular and behavioral processes in all vertebrate and invertebrate animals. The role played by peptide signals in information processing is fundamentally different from that of conventional neurotransmitters. Neuropeptides may act as neurotransmitters or neuromodulators and are released at either synaptic or non-synaptic sites. Peptide signals control developmental processes, drive specific behaviors or contribute to the mechanisms of learning and memory storage. Co-transmission within or across peptide families, and between peptide and non-peptide signaling molecules, is common; this ensures the great versatility of their action. How these tasks are fulfilled when multiple neuropeptides are released has become an important topic for peptide research. Although our knowledge concerning the physiological and behavioral roles of most of the neuropeptides isolated from molluscs is incomplete, this article provides examples to address the complexity of peptide signaling.

1. Voltage-clamp recordings from identified neurons of Helix pomatia L. revealed that two Ca-current components may contribute to the inward current in Na-free saline. 2. It was found that the transient component of the inward Ca-current was largely blocked by Ni 2+ but not by Cd 2+ . 3. CARP (10 −6 M) selectively suppressed LVA currents. This effect is also reflected by tail current measurements, where in control saline the deactivation could be fitted by two exponentials, while after CARP treatment, by one. 4. MIP (10 −6 M) specifically enhanced the LVA component, or shifted the I-V curve of the composite Ca-current in hyperpolarizing direction. 5. It is concluded that peptides (CARP and MIP) isolated from Mytilus ganglia selectively affect the LVA Ca-channels, and that this effect is supposed to be species-specific.