Anodonta cygnea, a freshwater swan mussel, exposed to diazinon: toxicity thresholds in behaviour and physiology.

Swan mussels (Anodonta cygnea) have been suggested as suitable bioindicators for the presence of pollutants in the environment. Application of the physiological and behavioral markers in these sessile species can be beneficial for environmental monitoring. The present study aimed to investigate the relationship between the behavioral disorders of movement and siphoning associated with the inhibition of tissue Acetylcholinesterase (AChE). For experiments, overally 120 bivalves of Anodonta cygnea (mean total length 80.33 ± 6.7 mm) were transported from the agricultural drains and canals in Sari county (Mazandaran Province, Iran) to our laboratory. First, the LC50-96 h of diazinon was estimated according to the Organization for Economic Co-operation and Development (OECD 1992) guideline with static water conditions. The sub-lethal toxicity pesticide experiments were conducted on the basis of the lowest observed effect concentration (LOEC) and the maximum acceptable toxicant concentration (MATC). The LC50-96 h, LOEC, and MATC values of diazinon were 85.2, 42.1, and 8.5 mg L- 1, respectively. Based on the observations of mussels' movement, the burrowing and displacement decreased with the concentration of toxicant in water. Moreover, the presence of diazinon in water and its exposure to experimental animals significantly reduces their siphoning rate. The RDA showed that the AChE activity had a higher correlation with the siphoning behavior than the movement behavior. The comparison of enzyme activity at different exposure and recovery times showed that there was a significant difference among the groups affected by the consumed pesticide (p = 0.001, between contrasts). The most remarkable morphometric characteristic was the siphon opening that was inversely correlated with the enzymatic activity. Studies in bioethics might benefit from paying attention to these traits that are directly related to the level of toxicity and behavioral adaptations required for animal survival.

The impact of iron coagulant on the behavior and biochemistry of freshwater mussels Anodonta cygnea and Unio tumidus during lake restoration.

Iron (Fe) treatment is one of the most commonly used methods to restore eutrophic lakes and reservoirs. The Fe-based coagulants dosage results in an almost immediate improvement in water quality at a relatively low cost. However, the effects of the application of coagulants are not always predictable, and the scale of the risks is not fully understood. The dosage of coagulants changes the chemical and physical properties of water, thereby affecting aquatic biocenoses. In this study, several laboratory experiments were conducted to evaluate the effects of Fe-based coagulant dosage on two bivalves species: Anodonta anatina and Unio tumidus. Their ability to efficiently filter water and reduce seston makes them a key component of aquatic ecosystems in terms of maintaining proper ecological health and stable functioning. Behavioral response, biochemical parameters, and body chemistry changes in mussels exposed to different doses of coagulant were surveyed. A dose-dependent reduction in filtration activity of both species was observed. As early as 10 g Fe m2 (which is a moderate dose used in lakes restoration), mussels of both species almost completely reduced their filtration activity and remained with closed valves for several subsequent days. Significant Fe accumulation in muscles of bivalves exposed to coagulant was also observed. This was particularly the case when very high doses of coagulant were applied. Then, the iron content in leg muscles of both species increased over fourfold. At the same time, a decrease in muscles calcium and phosphorus content was observed. No symptoms of oxidative stress (TBARS, H2O2) after mussels exposure to coagulants were found. The results suggest that the application of Fe-based coagulant for water ecosystem restoration may be a threat to the mussels population. These findings are significant for decisions on the selection of restoration methods for a specific lake.

Potential impacts of the invasive species Corbicula fluminea on the survival of glochidia.

Freshwater mussels (Bivalvia, Unionida) are one of the most imperilled faunal groups globally, being the introduction of invasive species a possible major mechanism of threat. The Asian clam Corbicula fluminea is a problematic invasive species in aquatic ecosystems and can impair the survival of parasitic larvae (glochidia) of native freshwater mussels. However, this possible mechanism of threat remains speculative and to date very few studies addressed quantitatively this issue. In order to cover this gap, we have performed a series of manipulative laboratory studies to assess how distinct densities of C. fluminea can affect the survival of glochidia after 6, 12, 24 and 48 h of exposure, using larvae of the native freshwater mussel Anodonta anatina. Our results suggest an increase in mortality of A. anatina glochidia with an increase in density of C. fluminea. Two main mechanisms may possibly explain our results: 1) the high filtration capacity of C. fluminea that can contribute to the mortality of glochidia due to the mechanical damage of their fragile shells when passing by siphons and/or digestive tract of C. fluminea and 2) the high excretion capacity of C. fluminea that can lead to mortality of glochidia due to increase in ammonia concentration. Mortality of glochidia was also time dependent with higher values registered after 48 h. This work is one of the first showing the influence of C. fluminea density on the survival of glochidia, being filtration (and consequent passage in the digestive tract) and biodeposition the main potential mechanisms explaining overall mortality. These results also suggest that sites with high densities of C. fluminea may be highly detrimental for the conservation of freshwater mussels, potentially impairing the survival of glochidia and negatively affecting the recruitment of juveniles.

Genotoxic, cytotoxic, and neurotoxic responses in Anodonta cygnea after complex metal mixture treatment.

Environmental effects associated with the release of various metals even at maximum permissible concentrations (MPC) to the aquatic ecosystems are evident. In the present work, time-dependent increase in accumulated metals amount in gills of Anodonta cygnea after exposure to complex metal (Zn 0.1, Cu 0.01, Ni 0.01, Cr 0.01, Pb 0.005, and Cd 0.005 mg/L, MPC accepted for the inland waters in EU) mixture at various time points (1, 2, 4, 7, 14, and 28 days) was investigated. Statistically significant increase of Cu and Cd was determined in mussel's gills after 7-day exposure, in comparison to control group; moreover, significantly elevated concentration of Cu was measured and after 14-day treatment (in comparison to control and pre-exposure group). Concentrations of five (Cu, Ni, Cr, Pb, and Cd) out of 6 investigated metals were statistically increased in gills tissue after 28-day treatment. Moreover, complex metal mixture has demonstrated tissue- and time-dependent genotoxicity (∑Gentox) and cytotoxicity (∑Cytox) responses in mussels. After 4-day exposure, there were found the highest ∑Gentox levels in gills cells and haemocytes. Two-day treatment of mussels resulted in the highest and statistically significant induction of ∑Cytox level (in gills). Furthermore, after short-term (4 days) exposure, statistically significant inhibition of AChE activity in hemolymph of metal mixture-exposed mussels, in comparison to control and pre-exposure group, was found. Comparison of investigated responses in different tissue of A. cygnea discloses new information about metal mixture (at MPC) impacts at different treatment time. According to the obtained geno- and cytotoxicity data, it is suggested that gills are more sensitive tissue. Environmentally relevant trace metal concentrations when existing in mixture are able to cause adverse effects in A. cygnea; therefore, biological effects at different levels of organism are expected as a realistic scenario.

Tissue-specific bioaccumulation, depuration and metabolism of 4,4'-dichlorodiphenyl sulfide in the freshwater mussel Anodonta woodiana.

Polychlorinated diphenyl sulfides (PCDPSs) are considered as a class of sulfur-containing dioxin-like pollutants with ubiquitous occurrence in natural waters and potential ecotoxicity to aquatic organisms. However, to date, no information is available regarding the bioaccumulation and biotransformation of PCDPSs in aquatic species. In this study, the uptake and depuration kinetics of 4,4'-dichlorodiphenyl sulfide (4,4'-di-CDPS) in the freshwater mussel Anodonta woodiana were investigated through semi-static exposure. The uptake rates (k1), depuration rates (k2), biological half-lives (t1/2) and tissue-specific bioconcentration factors (BCFs) of 4,4'-di-CDPS in the gill, liver and muscle were measured in the range of 0.509-21.734 L d-1 g-1 d.w., 0.083-0.221 d-1, 3.14-8.35 d and 3.662 × 103-124.979 × 103 L kg-1 l.w., respectively. With the increase in exposure dose, the values of k1 and BCFs were significantly reduced, indicating that low-dose exposure to 4,4'-di-CDPS could lead to more severe bioaccumulation. Based on the analysis of mass spectra of the extracted liver samples, the structures of four metabolites of 4,4'-di-CDPS were identified. Moreover, the levels of these metabolites were also quantitatively measured. The proposed metabolic pathways of 4,4'-di-CDPS in mussel liver included sulfur-oxidation, dechlorination and methoxylation. Comparatively, sulfur-oxidation was the predominant metabolic pathway of 4,4'-di-CDPS in the liver of A. woodiana. These results provide valuable data and fill the information gap on the bioaccumulation and metabolism of PCDPSs in freshwater species.

Freshwater mussels (Anodonta anatina) reduce transmission of a common fish trematode (eye fluke, Diplostomum pseudospathaceum).

Recent results suggest that bivalves can play an important role in restraining the spread of various aquatic infections. However, the ability of mussels to remove free-living stages of macroparasites and reduce their transmission is still understudied, especially for freshwater ecosystems. We investigated the influence of the common freshwater mussel (Anodonta anatina) on the transmission of a trematode (eye fluke, Diplostomum pseudospathaceum), which frequently infects fish in farms and natural habitats. In our experiments, mussels caused a significant decrease (P < 0·001) in the abundance of trematode free-living stages, from 6520 to 1770 cercariae L-1 on average (about 4-fold in 2 h). Individual clearance rates of mussels were 0·6‒3·7 L per hour (mean 1·9). These tests were followed by experimental infections of rainbow trout (Oncorhynchus mykiss) with different doses of D. pseudospathaceum cercariae in the presence or absence of mussels. Exposure of fish to cercariae in the presence of mussels significantly (P < 0·05) reduced the infection intensities in fish (by 30-40%) at all exposure doses. Our results indicate that freshwater bivalves can markedly reduce local cercariae densities and could be useful in mitigation of trematodoses harmful to fish farming.

Differences in tolerance to anthropogenic stress between invasive and native bivalves.

Tolerance towards environmental stress has been frequently considered as one of the key determinants of invasion success. However, empirical evidence supporting the assumption that invasive species can better endure unfavorable conditions compared with native species is limited and has yielded opposing results. In this study, we examined the tolerance to different stress conditions (thermal stress and trace metal zinc pollution stress) in two phylogenetically related and functionally similar freshwater bivalve species, the native Anodonta anatina and the invasive Sinanodonta woodiana. We assessed potential differences in response to stress conditions using several cellular response assays: efficiency of the multixenobiotic resistance mechanism, respiration estimate (INT reduction capacity), and enzymatic biomarkers. Our results demonstrated that the invasive species overall coped much better with unfavorable conditions. The higher tolerance of S. woodiana was evident from (i) significantly decreased Rhodamine B accumulation indicating more efficient multixenobiotic resistance mechanism; (ii) significantly higher INT reduction capacity and (iii) less pronounced alterations in the activity of stress-related enzymes (glutathione-S-transferase, catalase) and of a neurotoxicity biomarker (cholinesterase) in the majority of treatment conditions in both stress trials. Higher tolerance to thermal extremes may provide physiological benefit for further invasion success of S. woodiana in European freshwaters, especially in the context of climate change.

Establishing mussel behavior as a biomarker in ecotoxicology.

Most freshwater mussel species of the Unionoida are endangered, presenting a conservation issue as they are keystone species providing essential services for aquatic ecosystems. As filter feeders with limited mobility, mussels are highly susceptible to water pollution. Despite their exposure risk, mussels are underrepresented in standard ecotoxicological methods. This study aimed to demonstrate that mussel behavioral response to a chemical stressor is a suitable biomarker for the advancement of ecotoxicology methods that aids mussel conservation. Modern software and Hall sensor technology enabled mussel filtration behavior to be monitored real-time at very high resolution. With this technology, we present our method using Anodonta anatina and record their response to de-icing salt pollution. The experiment involved an environmentally relevant 'pulse-exposure' design simulating three subsequent inflow events. Three sublethal endpoints were investigated, Filtration Activity, Transition Frequency (number of changes from opened to closed, or vice versa) and Avoidance Behavior. The mussels presented a high variation in filtration behavior, behaving asynchronously. At environmentally relevant de-icing salt exposure scenarios, A. anatina behavior patterns were significantly affected. Treated mussels' Filtration Activity decreased during periods of very high and long de-icing salt exposure (p<0.001), however, increased during short de-icing salt exposure. Treated mussels' Transition Frequency increased during periods of very high and long de-icing salt exposure (p<0.001), which mirrored the Avoidance Behavior endpoint observed only by mussels under chemical stress. Characteristics of Avoidance Behavior were tighter shell closures with repeated and irregular shell movements which was significantly different to their undisturbed resting behavior (p<0.001). Additionally, we found that mussels were sensitive to a chemical stressor even when the mussel's valves were closed. Due to the effects of de-icing salt pollution on freshwater mussel behavior, we suggest better management practices for de-icing salt use be implemented. Our experimental method demonstrated that, with the application of current technologies, mussel behavioral response to a chemical stressor can be measured. The tested sublethal endpoints are suitable for mussel ecotoxicology studies. Avoidance Behavior proved to be a potentially suitable endpoint for calculating mussel behavior effect concentration. Therefore we recommend adult mussel behavior as a suitable biomarker for future ecotoxicological research. This method could be applied to other bivalve species and for physical and environmental stressors, such as particulate matter and temperature.

Population-specific responses to an invasive species.

Predicting the impacts of non-native species remains a challenge. As populations of a species are genetically and phenotypically variable, the impact of non-native species on local taxa could crucially depend on population-specific traits and adaptations of both native and non-native species. Bitterling fishes are brood parasites of unionid mussels and unionid mussels produce larvae that parasitize fishes. We used common garden experiments to measure three key elements in the bitterling-mussel association among two populations of an invasive mussel (Anodonta woodiana) and four populations of European bitterling (Rhodeus amarus). The impact of the invasive mussel varied between geographically distinct R. amarus lineages and between local populations within lineages. The capacity of parasitic larvae of the invasive mussel to exploit R. amarus was higher in a Danubian than in a Baltic R. amarus lineage and in allopatric than in sympatric R. amarus populations. Maladaptive oviposition by R. amarus into A. woodiana varied among populations, with significant population-specific consequences for R. amarus recruitment. We suggest that variation in coevolutionary states may predispose different populations to divergent responses. Given that coevolutionary relationships are ubiquitous, population-specific attributes of invasive and native populations may play a critical role in the outcome of invasion. We argue for a shift from a species-centred to population-centred perspective of the impacts of invasions.

Improvement of urban lake water quality by removal of Escherichia coli through the action of the bivalve Anodonta californiensis.

High levels of fecal indicator bacteria, such as Escherichia coli, can be indicative of poor water quality. The use of shellfish to reduce eutrophication has been proposed, but application of bivalves to reduce bacterial levels has not been extensively reported. Removal of E. coli by the native freshwater mussel Anodonta californiensis was studied using laboratory batch systems and field-based flow-through systems. Batch systems were utilized to determine the fate and inactivation of E. coli after uptake by the mussel. Batch experiments demonstrated that uptake patterns followed first order kinetics and E. coli was inactivated with less than 5% of the initial colonies recoverable in fecal matter or tissue. Flow-through systems located at an urban impaired lake in San Francisco, CA were utilized to determine uptake kinetics under environmentally relevant conditions. The bivalves maintained a 1-log removal of E. coli for the duration of exposure. The calculated uptake rates can be used in conjunction with hydrologic models to determine the number of bivalves needed to maintain removal of E. coli in different freshwater systems. The outcomes of this study support the use of native freshwater bivalves to achieve the co-benefits of rehabilitating a freshwater ecosystem and improving water quality via reduction of E. coli in contaminated freshwater systems.

Habitat pollution and thermal regime modify molecular stress responses to elevated temperature in freshwater mussels (Anodonta anatina: Unionidae).

Elevated temperature and pollution are common stressors in freshwater ecosystems. We study cellular stress response to acute warming in Anodonta anatina (Unionidae) from sites with different thermal regimes and pollution levels: a pristine area and an agriculturally polluted site with normal temperature regimes (F and A, respectively) and a polluted site with elevated temperature (N) from the cooling pond of an electrical power plant. Animals were exposed to different temperatures for 14 days and stress response markers were measured in gills, digestive gland and hemocytes. Mussels from site N and A had elevated background levels of lactate dehydrogenase activity indicating higher reliance on anaerobic metabolism for ATP production and/or redox maintenance. Exposure to 25°C and 30°C induced oxidative stress (indicated by elevated levels of lipid peroxidation products) in digestive gland and gills of mussels from A and F sites, while in mussels from N sites elevated oxidative stress was only apparent at 30°C. Temperature-induced changes in levels of antioxidants (superoxide dismutase, metallothioneins and glutathione) were tissue- and population-specific. Acute warming led to destabilization of lysosomal membranes and increased frequencies of nuclear lesions in mussels from F and A sites but not in their counterparts from N site. Elevated temperature led to an increase in the frequency of micronuclei in hemocytes in mussels from F and A sites at 25°C and 30°C and in mussels from N site at 30°C. The mussels from N site also demonstrated better survival at elevated temperature (30°C) than their counterparts from the F and A sites. Taken together, these data indicate that long-term acclimation and/or adaptation of A. anatina to elevated temperatures result in increased thermotolerance and alleviate stress response to moderate temperature rise. In contrast, extreme warming (30°C) is harmful to mussels from all populations indicating limit to this induced thermotolerance.

Seasonal changes in the behaviour and respiration physiology of the freshwater duck mussel, Anodonta anatina.

For low-energy organisms such as bivalves, the costs of thermal compensation of biological rates (synonymous with acclimation or acclimatization) may be higher than the benefits. We therefore conducted two experiments to examine the effect of seasonal temperature changes on behaviour and oxygen consumption. In the first experiment, we examined the effects of seasonal temperature changes on the freshwater bivalve Anodonta anatina, taking measurements each month for a year at the corresponding temperature for that time of year. There was no evidence for compensation of burrowing valve closure duration or frequency, or locomotory speed. In the second experiment, we compared A. anatina at summer and winter temperatures (24 and 4°C, respectively) and found no evidence for compensation of the burrowing rate, valve closure duration or frequency, or oxygen consumption rates during burrowing, immediately after valve closure or at rest. Within the experimental limits of this study, the evidence suggests that thermal compensation of biological rates is not a strategy employed by A. anatina. We argue that this is due to either a lack of evolutionary pressure to acclimatize, or evolutionary pressure to not acclimatize. Firstly, there is little incentive to increase metabolic rate to enhance predatory ability given that these are filter feeders. Secondly, maintained low energetic demand, enhanced at winter temperatures, is essential for predator avoidance, i.e. valve closure. Thus, we suggest that the costs of acclimatization outweigh the benefits in A. anatina.

Comparison of population genetic patterns in two widespread freshwater mussels with contrasting life histories in western North America.

We investigate population genetic structuring in Margaritifera falcata, a freshwater mussel native to western North America, across the majority of its geographical range. We find shallow rangewide genetic structure, strong population-level structuring and very low population diversity in this species, using both mitochondrial sequence and nuclear microsatellite data. We contrast these patterns with previous findings in another freshwater mussel species group (Anodonta californiensis/A. nuttalliana) occupying the same continental region and many of the same watersheds. We conclude that differences are likely caused by contrasting life history attributes between genera, particularly host fish requirements and hermaphroditism. Further, we demonstrate the occurrence of a 'hotspot' for genetic diversity in both groups of mussels, occurring in the vicinity of the lower Columbia River drainage. We suggest that stream hierarchy may be responsible for this pattern and may produce similar patterns in other widespread freshwater species.

[Regulation of adenylyl cyclase signaling system by insulin, biogenic amines, and glucagon at their separate and combined action in the muscle membranes of the mollusc Anodonta cygnea].

In smooth muscles of mollusc Anodonta cygnea, hormones produce regulatory effects on the adenylyl cyclase (AC) signaling system via receptors of the serpentine (biogenic amine, isoproterenol, glucagon) and of tyrosine kinase (insulin) types. Intracellular mechanisms of their action are interconnected. Use of hormones, their antagonists, and pertussis toxin at the combined action of insulin and biogenic amines or of glucagon on the AC activity allows revealing possible intersection points in mechanisms of their action. The combined effect of insulin and serotonin or of glucagon leads to a decrease of stimulation of AC by these hormones, whereas at action of insulin and isoproterenol the AC-stimulatory effect of insulin is blocked, while the AC-inhibitory effect of isoproterenol is preserved both in the presence and in the absence of the non-hydrolyzed GTP analog - guanylylimidodiphosphate (GppNHp). Specific blocking of the AC-stimulatory serotonin effect by cyproheptadine - an antagonist of serotonin receptors - did not affect stimulation of AC by insulin. Beta-adrenoblockers (propranolol and alprenolol) interfered with inhibition of the AC activity by isoproterenol, but did not change the AC stimulation by insulin. Pertussis toxin blocked the AC-inhibitory effect of isoproterenol and attenuated the AC-stimulatory effect of insulin. Thus, in muscles of the mollusc Anodonta cygnea there have been revealed negative interrelations between the AC system, which are realized at the combined effect of insulin and serotonin or of glucagon, probably at the level of receptor of the serpentine type (serotonin, glucagon), while at action of insulin and isoproterenol - at the level of interaction of G1 protein and AC.

Main partitioning criteria for the characterization of the health status in the freshwater mussel Anodonta cygnea from spontaneously polluted area in Western Ukraine.

The aim of this study was to appreciate the consequences of spontaneous human activity for freshwater mollusks in the generally ecologically sustainable area in Western Ukraine. For this, bivalve mollusk, Anodonta cygnea, at three sites, with mixed agricultural and municipal activities (A), close to a municipal water inlet (F) and the cooling pond of a nuclear power plant (N), were studied in spring, summer, and autumn. The set of parameters included the characteristics of oxidative stress (activity of catalase (CAT), levels of protein carbonyls (PC)), levels of reduced and oxidized glutathione (GSH, GSSG, respectively), activities of lactate dehydrogenase (LD), cholinesterase (ChE), ethoxyresorufin-O-deethylase (EROD), glutathione S-transferase (GST) in the digestive gland, and concentrations of vitellogenin-like proteins (Vtg-LP) in gonads and also morphological indices. Although the discriminant functional analysis confirmed the general seasonal regularities for studied groups, it allowed to discriminate between sites (P < 0.05). At site A, oxidative stress; high levels of LD, EROD, and GST; and low levels of ChE and condition factor were reflected. This demonstrated the sensitivity of mussels to constant effect of mixed pollution. At site N, oxidative injury was shown that might be explained by the constantly high temperature. At site F, abrupt elevations of Vtg-LP and EROD levels in autumn were probably related to an emergency situation on the nearby dump. So, both chronic and temporal environmental effects were reflected by a set of markers in mollusk. The classification and regression tree (CART) algorithm selected GSH and PC in the digestive gland and Vtg-LP as partitioning criteria for the characterization of mussel health status. © 2010 Wiley Periodicals, Inc. Environ Toxicol, 2012.

[Evaluation of biological effects of cobalt-nanocomposites with the use of biochemical markers of bivalve mollusk Anodonta cygnea].

Intensive implementation of nanomaterials requires development of novel methods for evaluation of their potential ecotoxicity. The aim of our study was to identify specific characteristics of the effect of cobalt-nanocomposite (Co-NC) on the molecular stress-responsive system in the digestive gland of bivalve mollusk Anodonta cygnea. Nanocomposite was synthesized by mixing alcohol solution of copolymer N-vinylpirrolidone, 5-(tret-butylperoxy)-5-methyl-1-hexene-3-yne and dimethylaminoethylmetacrylate and cobalt (II) chloride. After 14 days of the mollusk exposure in the presence of Co-NC, CoCl, or corresponding polymer substance it was shown that the Co-NC, in contrast to other agents, does not cause an oxidative stress due to the superoxide dismutase activity, metallotioneins (MTs) level, glutathione redox index and oxyradical production. Multivariate analysis confirmed specific features of the Co-NC's effect related to an enhanced expression of MTs, while CoCl2 activated lactate dehydrogenate and oxyradical production, and polymer substance enhanced glutathione transferase activity.

Seasonal variations of pH, pCO2, pO2, HCO3- and Ca2+ in the haemolymph: implications on the calcification physiology in Anodonta cygnea.

A study about the relationship between the physical-chemical parameters and the calcium carbonate balance between the haemolymph fluid and mantle calcareous structures was carried out in Anodonta cygnea. An intense peak of HCO(3) (-) and a highest pH in December-January months may be understood as a preparation period for creating alkaline conditions. An intense pH decrease from January to February in parallel with the HCO(3) (-) reduction seems to indicate the beginning process of carbonate precipitation. On the other hand, the following calcium and HCO(3) (-) increases in February-May associated with a continuous and gradual pH fall profile may infer two combined aspects: calcium and HCO(3) (-) absorption from external environment and a simultaneous intense calcium carbonate deposition in the haemolymph. So, the pCO(2) peak in this period reflects a subsequent result on equilibrium balance between HCO(3) (-) absorption and deposition. The only significant pO(2) increase in the next period, from February to June, is related with an energetic increase to support the metabolic activity favouring the posterior intense pCO(2) peaks. The extended time of CO(2) production in the haemolymph from May to November should induce an increased metabolic acidosis with subsequent intense formation of both HCO(3) (-) and Ca(2+) ions in the same period. This seems to result from CaCO(3) deposits dissolution in the haemolymph, the most direct calcareous source. Additionally, the later increase of metabolic succinic acid during autumn may greatly potentiate this acidosis increasing the dissolution process. Consequently, the pH profile present two simultaneous alkaline peaks in July and October, probably due to a strong HCO(3) (-) release from the CaCO(3) dissolution. So, the present seasonal results indicate that in the freshwater bivalve A. cygnea, the low metabolism with higher pH from the early winter is the main cause which may favour a calcareous precipitation, while the high metabolism with lower pH from the early summer may function as an inductor of calcareous dissolution in the haemolymph.

A mathematical model of the proton balance in the outer mantle epithelium of Anodonta cygnea L.

In the freshwater mollusc Anodonta cygnea and other unionids, the mantle plays an important role in the regulation of the movements of ions between the shell and the extrapaleal fluid. In this report, a mathematical model that attempts to describe the cell metabolic mechanisms underlying the operation of the outer mantle epithelium as a source of protons is presented. We encoded the information gathered by studying the epithelium in vitro, which includes the electrophysiology of the preparation, measurements of basic rates of transport of protons and base, the effect of metabolic and transport inhibitors on its electrical behavior and the dynamic measurements of pHi. The model was conceived so that the short-circuit current (Isc) and fluxes of Na+, K+ and Cl(-); intracellular volume; electrical potential; and ionic concentrations can be computed as a function of time. Furthermore, the analytical descriptions of all ionic fluxes involved are such that the effect of transport inhibitors can be simulated. In all the simulations performed, it was possible to reproduce the experimental results obtained with specific inhibitors of transport systems on the Isc and on pHi. In some cases, it was necessary to make alterations to one or more parameters of the reference condition. For each simulation carried out, the analysis of the results was consistent. The model is an analytical tool that can be used to show the internal coherence of the qualitative model previously proposed and to plan further experiments.

Potential role of cholinesterases in the invasive capacity of the freshwater bivalve, Anodonta woodiana (Bivalvia: Unionacea): a comparative study with the indigenous species of the genus, Anodonta sp.

To address the potential role of cholinesterase enzymes in the invasive capacity of species, the present study investigated ChE activity in the invasive freshwater bivalve Anodonta woodiana (Lea, 1834) comparing it with that of the indigenous species, Anodonta sp. (Linnaeus, 1758). The invasive capacity of pests has often been linked to their ecological plasticity and high intrinsic genetic variability; however the role played by molecular and cellular mechanisms, generally known as an organism's response to pollution, is unclear. Different substrates and selective ChE enzyme inhibitors were investigated in digestive gland, foot, gonad, adductor muscle and gill tissues while sensitivity to four organophosphate (OP) insecticides was investigated in vitro only in adductor muscle. The invasive species (A. woodiana) showed significantly greater (at least one order of magnitude) ChE activity than the endemic species (Anodonta sp.) (p<0.05) using acetylthiocholine (ASCh) as substrate and the activity was more widely distributed in tissues involved in movement (adductor muscle and foot), respiration, feeding (gills) and reproduction (gonads). Moreover, only the invasive species, A. woodiana, showed detectable ChE (vs. ASCh) activity in gill tissue. No substrate specificity was observed in any tissue of either species as already described for other bivalve species. ChE activity was not inhibited by Iso-OMPA but showed high sensitivity to BW248c51 and eserine. Both species showed moderate to low sensitivities in vitro to OP insecticides in the range 10(-7)-10(-2) M. Calculated IC(50) for fenitrothion and chlorpyrifos was in the range 10(-6)-10(-3) M in muscle of A. woodiana while a higher inhibition was observed for fenitrothion (10(-7) M) and lower for chlorpyrifos (10(-2) M) in the indigenous species Anodonta sp. Similar IC(50) of 10(-5)-10(-6) M were observed for DFP and azamethiphos in both species. The hypotheses of other authors that acetylcholinesterase (AChE) is involved in the control of many essential functions, such as frontal ciliary activity of gill epithelium, temperature resistance, ciliary activity for transport of suspended particulate, valve opening and embryo development, suggest that the high catalytic efficiency of the invasive species may endow it with a competitive advantage over the endemic species. In view of the peculiar reproductive strategy of these mussels, higher ChE vs. ASCh activity in gonads of the invasive species could also favour glochidium production and embryo development under a wider range of environmental conditions.

Effects of aqueous aluminium on four fish ectoparasites.

Effects of aqueous aluminium on four species of fish ectoparasites were studied. Fish infected by Gyrodactylus derjavini, G. macronychus and Anodonta anatina glochidia, and free living Argulus foliaceus were exposed to acidic Al-rich water (pH 5.8), acidic Al-poor water (pH 5.8) and control water (pH 6.3). The results showed that aqueous aluminium had a negative effect on the ectoparasites tested, without any apparent negative effect on the fish hosts. The effect of aluminium was dependent on the kind of organism tested. The gyrodactylids were eliminated, while the duck mussel glochidia showed a decrease in abundance when exposed to 200-260 mug Al/l at pH 5.8. The little fish louse showed increased mortality rate when exposed to aluminium. The effects of aluminium on the parasites in the present study confirm the importance of water quality in general, and acidification in particular, for the distribution and population dynamics of ectoparasites in freshwater environments.