Assessment of the joint effect of thermal stress,pollution, and parasitic infestation on the activity of antioxidative enzymes in pulmonate mollusk <Emphasis Type="Italic">Lymnaea stagnalis</Emphasis>

This study makes a multifactor analysis of the joint effect of reservoir contamination, parasitic infestation, and experimental hyperthermia on the activity of antioxidative enzymes (peroxidase, catalase, and glutathione S-transferase) in the pulmonate mollusk Lymnaea stagnalis from the clean Naroch Lake and contaminated Chizhovsky Reservoir in the Republic of Belarus. The activity of peroxidase is ascertained to be affected most strongly by the simultaneous effect of parasitic infestation and reservoir contamination and parasitic infestation coupled with experimental hyperthermia. The joint effect of parasitic infestation and reservoir pollution is found to influence the activity of catalase. Meanwhile, no correlation is found between the studied factors and the activity of glutathione S-transferase. It is shown that there is no mutual influence of the three studied factors on the activity of the investigated enzymes.     

Neuromodulatory effects of hydrogen peroxide on central neurons in the feeding network of the mollusc <Emphasis Type="Italic">Lymnaea stagnalis</Emphasis>

Hydrogen peroxide at a concentration of 100 μM was found to exert a pronounced modulatory effect on motor (R/L cells in B1–B4 clusters) and modulatory (R/L cerebral giant cells) neurons in the feeding neural network of the mollusc Lymnaea stagnalis as manifested in changes in the firing rate, membrane potential level and spike amplitude in these cells. The observed effects were reversible, transient, and reached their peak values in 1 min since application of the preparation. Injection of hydrogen peroxide into the cavity of the cephalopedal sinus resulted in no statistically significant changes in the parameters of mollusc feeding behavior. Hydrogen peroxide is assumed to act as a rapid neuromodulator towards neurons of the central feeding rhythm generator in Lymnaea stagnalis.     

Responses of the antioxidative and biotransformation enzymes in the aquatic fungus <Emphasis Type="Italic">Mucor hiemalis</Emphasis> exposed to cyanotoxins

Objectives

To investigate antioxidative and biotransformation enzyme responses in Mucor hiemalis towards cyanotoxins considering its use in mycoremediation applications.

Results

Catalase (CAT), glutathione reductase (GR), and glutathione peroxidase (GPx) in M. hiemalis maintained their activities at all tested microcystin-LR (MC-LR) exposure concentrations. Cytosolic glutathione S-transferase (GST) activity decreased with exposure to 100 µg MC-LR l^?1 while microsomal GST remained constant. Cylindrospermopsin (CYN) at 100 µg l^?1 led to an increase in CAT activity and inhibition of GR, as well as to a concentration-dependent GPx inhibition. Microsomal GST was inhibited at all concentrations tested. β-N-methylamino-l-alanine (BMAA) inhibited GR activity in a concentration-dependent manner, however, CAT, GPx, and GST remained unaffected.

Conclusions

M. hiemalis showed enhanced oxidative stress tolerance and intact biotransformation enzyme activity towards MC-LR and BMAA in comparison to CYN, confirming its applicability in bioreactor technology in terms of viability and survival in their presence.

     

Two Subtypes of Nicotinic Acetylcholine Receptors in <Emphasis Type="Italic">Lymnaea stagnalis</Emphasis> Neurons Control Chloride Conductance

Giant neurons of the mollusc Lymnaea stagnalis contain heterogeneous population of nicotinic acetylcholine receptors (nAChRs) according to their relative sensitivity to antagonists. All these receptors are involved in the total response to acetylcholine (ACh). To evaluate activity of different pharmacological agents correctly it is necessary to know ionic selectivity of nAChRs which participate in transmembrane ionic current. In this work we studied the influence of ionic composition of the external and intracellular solutions on the current amplitude and current–voltage relation under the action of ACh or other nAChR agonists on the identified neurons of the left and right parietal ganglia of Lymnaea. After non-permeable cation N-methyl-D-glucamine was completely substituted for external Na⁺ ions there were no changes in the current characteristics. After a 10-fold decrease in Cl–concentration in the external solution there was a considerable shift of the current–voltage curve to the right, outward currents at the holding potential (V_h) up to 30 mV were not observed. On the contrary, a 10-fold decrease of Cl^– concentration in the intracellular solution led to a shift of the current–voltage curve to hyperpolarizing direction, the reversal potential shift was in the average –42 mV. When ACh and nicotinic agonists with higher selectivity towards vertebrate α7 neuronal nAChR type and one of the two subtypes of Lymnaea nAChRs were compared, no differences in changes of ionic current characteristics were found. Neurons with distinct relative fraction of one or another nAChR subtype reacted to Cl^– concentration change in the same way. Our results support earlier data on Cl^– mechanism of Lymnaea neuron responses to ACh and evidence identical ionic selectivity of the two nAChR subtypes in identified neurons tested.     

Food web interactions determine energy transfer efficiency and top consumer responses to inputs of dissolved organic carbon

The sand stored in the gizzard of some freshwater snails should assist in the mechanical digestion of tough food, yet effects of gizzard sand on consumption of living macrophyte tissue are seldom addressed. We quantified the effects of gizzard sand both on consumption of plant tissue and on snail growth and reproduction with a cross-gradient, 28-day laboratory experiment. We offered apical shoot sections of Myriophyllum spicatum (initial ash content ~ 56%) or soft lettuce (ash content ~ 19%, as control) to Lymnaea stagnalis previously reared on sand or without sand. Gizzard sand had no effect on snail growth but sand-reared snails fed with lettuce initially produced more eggs than their no-sand counterparts. Snails fed M. spicatum exhibited stunted growth and produced only one small egg clutch. Shell height at time of first oviposition of M. spicatum-fed snails was two-thirds of that of lettuce-fed controls. Snails initially appeared to feed on periphyton and older, brownish M. spicatum leaflet tips, but later turned to midstem tissue. Fresh apices were not consumed. Gizzard sand had a limited effect on the ability of L. stagnalis to consume living M. spicatum, with apices likely defended chemically (polyphenols) and midstem tissue defended structurally (high ash content).     

New paradoxical three-finger toxin from the cobra <Emphasis Type="Italic">Naja kaouthia</Emphasis> venom: Isolation and characterization

A new three-finger toxin nakoroxin was isolated from the cobra Naja kaouthia venom, and its complete amino acid sequence was established. Nakoroxin belongs to the group of “orphan” toxins, data on the biological activity of which are practically absent. Nakoroxin shows no cytotoxicity and does not inhibit the binding of α-bungarotoxin to nicotinic acetylcholine receptors of muscle and α7 types. However, it potentiates the binding of α-bungarotoxin to the acetylcholine-binding protein from Lymnaea stagnalis. This is the first toxin with such an unusual property.     

Antioxidative protection in the central nervous ganglia of a mollusc Lymnaea stagnalis at modulation of activity of the NO-ergic system

In experiments on molluscs Lymnnaea stagnalis the state of antioxidative protection is studied in central nervous ganglia during a long-term activation (inhibition) of synthesis of nitric monoxide (NO) in the body. Effect of the blocker of NO-synthase N(G)-nitro-L-arginine (L-NNA) at the background of enhancement of pulmonary respiration has been established to be associated with a rise of levels of reduced glutathione and TBK-active products in the nervous tissue at preservation of a relatively high superoxide dismutase activity and a low glutathione peroxidase activity as compared with control group and the animals treated with the metabolic precursor of NO synthesis L-arginine. In spite of the revealed disturbances of balance of the body pro- and antioxidative system, DNA electrophoresis detected no products of its degradation, which can indicate the absence of massive programmed death of the nervous tissue cells in Lymnaea stagnalis during modulation of activity of the NO-ergic system.     

Resistance to herbivory of two populations of <Emphasis Type="Italic">Elodea</Emphasis><Emphasis Type="Italic">canadensis</Emphasis> Michaux and <Emphasis Type="Italic">Elodea nuttallii</Emphasis> Planchon St. John

In this article, we compared the resistance of two introduced populations of Elodea nuttallii and Elodea canadensis to two different herbivores. Samples were collected from the River Rhine and River Rhône in eastern France. The two populations of E. nuttallii differed in their introduction history, whereas E. canadensis was introduced at the same time in the two sites. The Daily Food Consumption (DFC) rates of the two macrophyte populations were evaluated in no-choice experiments using the scraper Lymnaea stagnalis and the shredder Gammarus roeseli. At the same time, we assessed four plant traits: dry matter content (DMC), total nitrogen content, carbon/nitrogen ratio and total phenolic content. The two populations of E. canadensis were consumed at low levels by both the herbivores. L. stagnalis showed a higher DFC on the Rhône population of E. nuttallii than on the Rhine population. No significant difference between the two populations was established with G. roeseli, but the level of DFC was high. This result demonstrates that the assessment of plant palatability should be carried out with several generalist herbivores belonging to various feeding groups (e.g. scrapers or shredders). Although the Rhône population of E. nuttallii had higher levels of phenols than the other populations, it was consistently consumed in greater quantities than E. canadensis. Neither the phenolic contents were not effective against these herbivores, nor the levels of phenolics too low to induce an efficient resistance. The higher DMC and the lower DFC of the two populations of E. canadensis suggest that this introduced plant has co-evolved with indigenous enemies in the introduced range.     

Iron homeostasis in tropical <Emphasis Type="Italic">indica</Emphasis> rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) cultivars having contrasting grain iron concentration

Iron homeostasis was studied in two tropical indica rice cultivars viz. Sharbati (high Fe) and Lalat (low Fe) having contrasting grain Fe concentration. Plants were hydroponically grown with 5 concentrations of Fe (0.05, 2, 5, 15, 50 mg L^?1) till maturity. The effect of incremental Fe treatment on the plant was followed by analyzing accumulation of ferritin protein, activities of aconitase enzyme, enzymes of anti-oxidative defense and accumulation of hydrogen peroxide and ascorbic acid. Plant growth was adversely affected beyond 15 mg L^?1 of Fe supplementation and effects of Fe stress (both deficiency and excess) were more apparent on the high Fe containing cultivar Sharbati than the low Fe containing Lalat. Level of ferritin protein and aconitase activity increased up to 5 mg L^?1 of Fe concentration. Lalat continued to synthesize ferritin protein at much higher Fe level than Sharbati and the cultivar also had higher activities of peroxidase, superoxide dismutase and glutathione reductase. It was concluded that the tolerance of Lalat to Fe stress was because of its higher intrinsic ability to scavenge free radicals of oxidative stress for possessing higher activity of antioxidative enzymes. This, together with its capacity to sequester the excess Fe in ferritin protein over a wider range of Fe concentrations made it more tolerant to Fe stress.     

The role of thiol redox systems in the peroxide stress response of <Emphasis Type="Italic">Escherichia coli</Emphasis>

The effect of mutations in the genes encoding glutathione, glutaredoxin, thioredoxin, and thioredoxin reductase on the response of growing Escherichia coli to oxidative stress was studied. The gshA mutants defective in glutathione synthesis had the lowest resistance to high doses of H₂O₂, whereas the trxB mutants defective in thioredoxin reductase synthesis had the highest resistance to this oxidant, exceeding that of the parent strain. Among the studied mutants, the trxB cells demonstrated the highest basic levels of catalase activity and intracellular glutathione; they were able to rapidly reach the normal GSH level after oxidative stress. At the same time, these bacteria showed high frequency of induced mutations. The expression of the katG and sulA genes suggests that, having different sensitivity to high oxidant concentrations, the studied mutants differ primarily in their ability to induce the antioxidant genes of the OxyR and SOS regulons.     

Effects of cadmium and lead stress on somatic embryogenesis of coniferous species. Part II: Changes of thiol substances

Conifers are often used as biomarkers of industrial pollution; however, little is known about the effects of heavy metals on them because only a few species have been tested. The aim of this work was to investigate the effects of cadmium (Cd²⁺) and lead (Pb²⁺) at three different concentrations (50, 250, and 500 µM) on the detoxification potential of Abies alba and Picea abies embryogenic cell masses throughout the 21-day proliferation period. Embryogenic cell masses of A. alba and P. abies responded to treatment with cadmium and lead by inducing phytochelatins and their biosynthetic intermediates. With increasing heavy metal concentrations, glutathione was used for the synthesis of phytochelatins enabling the tissues to bind to heavy metal ions and thereby avoiding the production of reactive oxygen species. Lead in A. alba and cadmium in both species caused similar increases of all antioxidative thiol compounds; thus, similar mechanisms involving a heavy metal-induced stress response can be assumed. In P. abies, the lowest lead concentration tested provoked the highest antioxidative response. Since a very low uptake of lead into the tissue was observed, the higher resistance of P. abies can be attributed to its ability to reduce lead uptake after longer exposure times. The results of cadmium treatment of both species and lead treatment of A. alba indicated the possibility of testing these coniferous species as potential phytoremediators. This is the first study to analyze the effects of heavy metals on the low-molecular-weight plant thiol content in A. alba embryogenic cell masses.     

Larval identification of two syntopic species <Emphasis Type="Italic">Branchipus schaefferi</Emphasis> and <Emphasis Type="Italic">Tanymastix stagnalis</Emphasis> (Crustacea,Anostraca) sheds light on their pattern of coexistence

In the present study we applied a discriminant function analysis to distinguish two fairy shrimp species, Branchipus schaefferi and Tanymastix stagnalis (Anostraca), co-occurring in Lago dell’Orso in central Italy. Our aim was to obtain information on the pattern of coexistence of these two species to investigate their syntopy since early larval stages. To identify the larvae of the two species we used six discriminant functions, one for each larval stage considered (nauplius, four metanauplius stages and juvenis), and based on morphometric analyses and measurements of different traits (thoracic appendages, body length, length of first antenna, length of head, thorax length and length of furca). The pool was visited at weekly intervals during two time periods for a total of 4 years (1995–1996 and 2000–2001) and physical and chemical variables (water level, water and air temperature, pH and conductivity) were recorded. A total of 1067 specimens were collected, measured and morphometrically identified. B. schaefferi was more abundant and persistent than T. stagnalis. At any stage, T. stagnalis was bigger than B. schaefferi and this difference may justify niche differentiation between coexisting species. Difference in size might be linked to difference in life-span as B. schaefferi reproduced earlier and at a smaller size than T. stagnalis. When the hydroperiod is short, B. schaefferi might be the only species to produce resting eggs in time for reproduction. On the other hand, T. stagnalis may have an advantage in the longest hydroperiods. In an ephemeral seasonal pond such as Lago dell’Orso, coexistence can be fostered and competitive exclusion can be prevented as a result of seasonal environmental changes.     

Glutathione improves survival of cryopreserved embryogenic calli of <Emphasis Type="Italic">Agapanthus praecox</Emphasis> subsp. <Emphasis Type="Italic">orientalis</Emphasis>

The effect of supplementation of reduced glutathione (GSH) to cryoprotectant solution on the generation of reactive oxygen species (ROS) (e.g., H₂O₂, OH^·, and O ₂ ^·? ) and antioxidants (e.g., SOD, POD, CAT, AsA, and GSH), as well as membrane lipid peroxidation (i.e., MDA content) mitigation in cryopreserving of embryogenic calli (EC) of Agapanthus praecox subsp. orientalis was investigated. The vitrification-based cryopreservation method was used in this study. The addition of GSH at a final concentration of 0.08 mM to the cryoprotectant solution has significantly improved cryotolerance of A. praecox EC. The EC post-thaw survival rate increased by 68.34 % using the cryoprotectant solution containing 0.08 mM GSH as compared to the control (GSH-free). EC treated with GSH displayed the reduction in  OH^· generation activity and the contents of H₂O₂ and MDA, as well as enhancement in the inhibition of O ₂ ^·? generation and the antioxidant activity. Treatment with exogenous GSH also increased endogenous AsA and GSH contents after dehydration step. Expression of stress-responsive genes, e.g., peroxidase (POD), peroxiredoxin, ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR), and glutathione peroxidase (GPX), was also increased during cryopreservation processes. The expression of DAD1 (Defender against apoptotic cell death) was elevated, while cell death-related protease SBT was suppressed. These results demonstrated that the addition of GSH to cryoprotectant solution affects the ROS level and could effectively improve survival of A. praecox EC through enhancing antioxidant enzyme activities and decreasing cell death.     

Two distinct <Emphasis Type="Italic">waxy</Emphasis> alleles impact the granule-bound starch synthase in sorghum

The granule-bound starch synthase (GBSS) is the enzyme responsible for amylose synthesis in starch granules. Loss of GBSS activity results in starch granules containing mostly amylopectin and little or no amylose, a phenotype described as waxy. Previously, two phenotypic classes of waxy alleles were identified in sorghum (Sorghum bicolor L. Moench) characterized by the absence (waxy ^a ; wx ^a ) or presence (waxy ^b ; wx ^b ) of the GBSS protein in the endosperm. To characterize these alleles, we examined endosperm architecture using scanning electron microscopy (SEM), assayed GBSS enzymatic activities, and identified DNA lesions associated with the mutations in the GBSS (Sb10g002140) gene. wx ^a , the allele present in B Tx630 and R Tx2907, contained a large insertion in the third exon, which was consistent with the absence of the GBSS protein previously observed. wx ^b , the allele present in B 9307 and B TxARG1, contained a missense mutation that resulted in conversion of glutamine 268 to histidine in a conserved domain in starch synthases. In wx ^b , GBSS activity was less than 25% that of the non-waxy line B Wheatland, and GBSS activity was not detected in wx ^a . SEM showed that endosperm architecture was very similar in both wx ^a and wx ^b alleles, but altered in comparison to non-waxy lines R Tx430 and B Wheatland. Both alleles may have a range of potential applications in grain sorghum because of low amylose content in their starch and the presence or absence of the GBSS protein. PCR based markers were developed for both the wx ^a and the wx ^b alleles to aid in molecular breeding of low amylose sorghum.     

Role of chemical signalling in release of motor programs during embryogenesis of freshwater snails <Emphasis Type="Italic">Lymnaea stagnalis</Emphasis> and <Emphasis Type="Italic">Helisoma trivolvis</Emphasis>

We have earlier found that freshwater pond snails Helisoma trivolvis and Lymnaea stagnalis, when reared under conditions of starvation, release chemical signals that reversibly suppress larval development of conspecific embryos. Here, we report that (i) these signals are not strictly conspecific and affect also the embryos of a closely related species, which occupies a similar environmental niche; (ii) besides the development of embryos, the signals also affect the release of main motor programs, such as locomotion, feeding, and cardiac activity; (iii) action of the signals is bidirectional: they retard the development and release of motor programs at the early larval stages (trochophore to veliger) and accelerate them at later stages (late veliger to hatching). A possible adaptive significance of the described phenomena is discussed.     

Interactions Between the Circadian Clock and Heme Oxygenase in the Retina of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

The Drosophila retina has an autonomous peripheral circadian clock in which the expression of the gene encoding heme oxygenase (HO) is under circadian control with the ho mRNA peaking at the beginning of the day and in the middle of the night. The function of HO in the retina is unknown, but we observed that it regulates the circadian clock and protects photoreceptors against DNA damage. The decline in HO level increases and decreases the expression of the canonical clock genes period (per) and Clock (Clk), respectively. The opposite result was observed after increasing HO expression. Among three products of HO activity—carbon monoxide (CO), ferrous ions, and biliverdin—the latter has no effect on per and Clk expressions, but CO exerts the same effect as the increase of ho expression. This suggests that HO action on the clock is mediated by CO, which may affect Clk expression during the day and the level of per expression. While ho expression is not stimulated by nitric oxide (NO), NO has the same effect on the clock as HO, increasing Clk expression and decreasing the expression of per.     

Histological evidence that diploid hybrids of <Emphasis Type="Italic">Cobitis taenia</Emphasis> and <Emphasis Type="Italic">C. elongatoides</Emphasis> (Teleostei,Cobitidae) develop into fertile females and sterile males

Rising temperatures likely affect the trophic interactions in temperate regions as global warming progresses. An open question is how a temperature rise may affect consumer pressure and plant abundance in shallow aquatic ecosystems, where most consumers are omnivorous. Interestingly, herbivory (plant-eating) is more prevalent toward low latitudes in ectotherms such as fish and aquatic invertebrates, and this may be temperature driven. We used pond snails (Lymnaea stagnalis L.) as a model aquatic ectotherm species and tested their consumption of both animal prey (Gammarus pulex L.) and plant material (Potamogeton lucens L.) at three different temperatures (15, 20, and 25°C). Higher temperatures led to higher consumption rates by the omnivore on both plant food and animal prey when fed separately. When the food was offered simultaneously, the pond snails consistently preferred animal prey over plant material at all tested temperatures. However, the omnivore did consume plant material even though they had enough animal prey available to them. Based on our experiments, we conclude that with increasing temperatures, L. stagnalis will only increase their consumption rates but not change food preference. Further studies are needed to test the generality of our findings across aquatic species to predict the effect of warming on aquatic plant consumption.     

Regulation of Multimeric Structure of NO-synthase as a New Factor of Organogenesis

The effect of NO on organogenesis in Drosophila is discussed. A new model of regulation of the activity of NO-producing enzyme, NO synthase is described, which takes into account endogenous synthesis of its reduced isoform. The reduced isoform of NO synthase is capable of suppressing the enzymatic activity of full-sized NO synthase during formation of a heterodimer in vivo and in vitro. The reduced form of this enzyme inhibits the antiproliferative effect of the full-sized NO synthase isoform during formation of eye structure in Drosophila by affecting the pathways of cell cycle regulation. The reduced form of NO synthase is an endogenous dominant-negative factor of regulation of the NO synthase activity in development of Drosophila.