Energy status of chironomid larvae (Diptera: Chironomidae) from high alpine rivers (Tyrol, Austria).

Chironomids (non-biting midges) inhabit almost every wet or semi-wet continental environment on Earth with probably 10,000 different species. Species occurrence and composition are undoubtedly limited by environmental harshness and food availability being reflected in their energy stores. Most animals store energy as glycogen and lipid. They enable the animals to survive adverse situations and to continue growth, development, and reproduction. This general statement is also true for insects and also particularly true for chironomid larvae. The rationale behind this research was, that probably any stress, any environmental burden, and any harmful influence increases the energy requirement of individual larvae depleting energy stores. We developed new methods to measure the glycogen and lipid content in small tissue samples. Here we show how to apply these methods to single chironomid larvae to demonstrate their energy stores. We compared different locations of the high Alpine rivers along harshness gradient densely populated and dominated by chironomid larvae. All samples show very low energy stores without any major differences. We found glycogen concentrations below 0.01% of dry weight (DW) and lipid concentrations below 5% of DW irrespective of the specific sampling point. These values are among the lowest ever observed in chironomid larvae. We demonstrate that individuals living in extreme environment are stressed leading to reduced energy stores in their bodies. This appears to be a general feature of high altitude regions. Our results provide new insights and a better understanding of population and ecological dynamics in harsh mountainous areas, also in view of a changing climate.

The dynamics of band (peak) shape development in capillary zone electrophoresis in the case of two co-migrating analytes: The displacement and the tag-along effects.

Peak shapes in electrophoresis are often distorted from the ideal Gaussian shape due to disturbing phenomena, of which the most important is electromigration dispersion. For fully dissociated analytes, there is a tight analogy between nonlinear models describing a separation process in chromatography and electrophoresis. When the velocity of the separated analyte depends on the concentration of the co-analyte, the consequence is a mutual influence of the analytes couples, which distorts both analyte zones. In this paper, we introduce a nonlinear model of electromigration for the analysis of two co-migrating fully dissociated analytes. In the initial stages of separation, they influence each other, which causes much more complicated peak shapes. The analysis has revealed that the two most important phenomena-the displacement and the tag-along effects-are common both for nonlinear chromatography and electrophoresis, though their description is partly based on rather different phenomena. The comparison between the nonlinear model of electromigration we describe and the numerical computer solution of the original continuity equations has proven an almost perfect agreement. The predicted features in peak shapes in initial stages of separation have been fully confirmed by the experiments.

Cadmium Uptake, MT Gene Activation and Structure of Large-Sized Multi-Domain Metallothioneins in the Terrestrial Door Snail Alinda biplicata (Gastropoda, Clausiliidae).

Terrestrial snails (Gastropoda) possess Cd-selective metallothioneins (CdMTs) that inactivate Cd2+ with high affinity. Most of these MTs are small Cysteine-rich proteins that bind 6 Cd2+ equivalents within two distinct metal-binding domains, with a binding stoichiometry of 3 Cd2+ ions per domain. Recently, unusually large, so-called multi-domain MTs (md-MTs) were discovered in the terrestrial door snail Alinda biplicata (A.b.). The aim of this study is to evaluate the ability of A.b. to cope with Cd stress and the potential involvement of md-MTs in its detoxification. Snails were exposed to increasing Cd concentrations, and Cd-tissue concentrations were quantified. The gene structure of two md-MTs (9md-MT and 10md-MT) was characterized, and the impact of Cd exposure on MT gene transcription was quantified via qRT PCR. A.b. efficiently accumulates Cd at moderately elevated concentrations in the feed, but avoids food uptake at excessively high Cd levels. The structure and expression of the long md-MT genes of A.b. were characterized. Although both genes are intronless, they are still transcribed, being significantly upregulated upon Cd exposure. Overall, our results contribute new knowledge regarding the metal handling of Alinda biplicata in particular, and the potential role of md-MTs in Cd detoxification of terrestrial snails, in general.

The dynamics of band (peak) shape development in capillary zone electrophoresis in light of the linear theory of electromigration.

The continuity equations that describe the movement of ions in liquid solutions under the influence of an external stationary electric field, as it is utilized in electrophoresis, were introduced a long time ago starting with Kohlrausch in 1897. From that time on, there have been many attempts to solve the equations and to discuss the results. In electrophoresis, special attention has always been devoted to the peak shapes obtained by the detector since the shapes have a tight connection with the phenomena taking place during electromigration and influence the efficiency and selectivity of the separation. Among these phenomena, the most important is electromigration dispersion. In this commented review paper, we compare various models of electromigration, try to find points that connect them, and discuss the range of their validity in light of the linear and nonlinear theory of electromigration.

Generalized model of the linear theory of electromigration and its application to electrokinetic chromatography: Theory and software PeakMaster 6-Next Generation.

The linear theory of electromigration, including the first-order nonlinear approximation, is generalized to systems with any equilibria fast enough to be considered instantaneous in comparison with the timescale of peak movement. For example, this theory is practically applied in the electrokinetic chromatography (EKC) mode of the CZE. The model enables the calculation of positions and shapes of analyte and system peaks without restricting the number of selectors, the complexation stoichiometry, or simultaneous acid-base equilibria. The latest version of our PeakMaster software, PeakMaster 6-Next Generation, implements the theory in a user-friendly way. It is a free and open-source software that performs all calculations and shows the properties of the background electrolyte and the expected electropherogram within a few seconds. In this paper, we mathematically derive the model, discuss its applicability to EKC systems, and introduce the PeakMaster 6 software.

Cadmium Pathways in Snails Follow a Complementary Strategy between Metallothionein Detoxification and Auxiliary Inactivation by Phytochelatins.

Metal detoxification is crucial for animals to cope with environmental exposure. In snails, a pivotal role in protection against cadmium (Cd) is attributed to metallothioneins (MTs). Some gastropod species express, in a lineage-specific manner, Cd-selective MTs devoted exclusively to the binding and detoxification of this single metal, whereas other species of snails possess non-selective MTs, but still show a high tolerance against Cd. An explanation for this may be that invertebrates and in particular snails may also synthetize phytochelatins (PCs), originally known to be produced by plants, to provide protection against metal or metalloid toxicity. Here we demonstrate that despite the fact that similar mechanisms for Cd inactivation exist in snail species through binding of the metal to MTs, the actual detoxification pathways for this metal may follow different traits in a species-specific manner. In particular, this depends on the detoxification capacity of MTs due to their Cd-selective or non-specific binding features. In the terrestrial slug Arion vulgaris, for example, Cd is solely detoxified by a Cd-selective MT isoform (AvMT1). In contrast, the freshwater snail Biomphalaria glabrata activates an additional pathway for metal inactivation by synthesizing phytochelatins, which compensate for the insufficient capacity of its non-selective MT system to detoxify Cd. We hypothesize that in other snails and invertebrate species, too, an alternative inactivation of the metal by PCs may occur, if their MT system is not Cd-selective enough, or its Cd loading capacity is exhausted.

Challenging the Metallothionein (MT) Gene of Biomphalaria glabrata: Unexpected Response Patterns Due to Cadmium Exposure and Temperature Stress.

Metallothioneins (MTs) are low-molecular-mass, cysteine-rich, metal binding proteins. In most animal species, they are involved in metal homeostasis and detoxification, and provide protection from oxidative stress. Gastropod MTs are highly diversified, exhibiting unique features and adaptations like metal specificity and multiplications of their metal binding domains. Here, we show that the MT gene of Biomphalaria glabrata, one of the largest MT genes identified so far, is composed in a unique way. The encoding for an MT protein has a three-domain structure and a C-terminal, Cys-rich extension. Using a bioinformatic approach involving structural and in silico analysis of putative transcription factor binding sites (TFBs), we found that this MT gene consists of five exons and four introns. It exhibits a regulatory promoter region containing three metal-responsive elements (MREs) and several TFBs with putative involvement in environmental stress response, and regulation of gene expression. Quantitative real-time polymerase chain reaction (qRT-PCR) data indicate that the MT gene is not inducible by cadmium (Cd) nor by temperature challenges (heat and cold), despite significant Cd uptake within the midgut gland and the high Cd tolerance of metal-exposed snails.

Affinity capillary electrophoresis: the theory of electromigration.

We focus on the state-of-the-art theory of electromigration under single and multiple complexation equilibrium. Only 1:1 complexation stoichiometry is discussed because of its unique status in the field of affinity capillary electrophoresis (ACE). First, we summarize the formulas for the effective mobility in various ACE systems as they appeared since the pioneering days in 1992 up to the most recent theories till 2015. Disturbing phenomena that do not alter the mobility of the analyte directly but cause an unexpected peak broadening have been studied only recently and are also discussed in this paper. Second, we turn our attention to the viscosity effects in ACE. Change in the background electrolyte viscosity is unavoidable in ACE but numerous observations scattered throughout the literature have not been reviewed previously. This leads to an uncritical employment of correction factors that may or may not be appropriate in practice. Finally, we consider the ionic strength effects in ACE, too. Limitations of the current theories are also discussed and the tasks identified where open problems still prevail. Graphical Abstract A weak base (A) undergoes an acidic-basic equilibria (in blue) and migrates with an electrophoretic mobility of [Formula: see text]. Simultaneously, it interacts with a selector (sel) while the analyte-selector complex migrates with an electrophoretic mobility of [Formula: see text]. The strength of the interaction (in orange) is governed by the binding constant, K A , and the concentration of the selector, c sel . This all gives the analyte an effective mobility of [Formula: see text] and moves it out of the zero position (EOF; right top insert). The interaction of the positively charged analyte with the neutral selector slows down the analyte with increasing selector concentration (right bottom insert).

Determination of the correct migration time and other parameters of the Haarhoff-van der Linde function from the peak geometry characteristics.

For Gaussian peaks, the migration time of the analyte results as the position of the top of the peak and the zone variance is proportional to the peak width. Similar relations have not yet been derived for the Haarhoff-van der Linde (HVL) function, which appears as a fundamental peak-shape function in electrophoresis. We derive the relations between the geometrical measures of the HVL-shaped peak, that is the position of its maximum, its width and a measure of its asymmetry, and the respective parameters a1, a2, and a3, of the corresponding HVL function. Under the condition of the HVL-shaped peak, the a1 parameter reflects the true migration time of the analyte, which may differ from the peak top position significantly. Our procedure allows us to express the parameters without the need of any external data processing (nonlinear regression). We demonstrate our approach on simulated peaks and on experimental data integrated by the ChemStation software (delivered with the CE instrumentation by Agilent Technologies). A significant improvement is achieved reading the migration time of the experimental and simulated peaks, which draws the error of the HVL-shaped peak migration time evaluation down to the resolution of the data sampling rate.

Equivalent peak resolution: characterization of the extent of separation for two components based on their relative peak overlap.

Although the classical formula of peak resolution was derived to characterize the extent of separation only for Gaussian peaks of equal areas, it is often used even when the peaks follow non-Gaussian distributions and/or have unequal areas. This practice can result in misleading information about the extent of separation in terms of the severity of peak overlap. We propose here the use of the equivalent peak resolution value, a term based on relative peak overlap, to characterize the extent of separation that had been achieved. The definition of equivalent peak resolution is not constrained either by the form(s) of the concentration distribution function(s) of the peaks (Gaussian or non-Gaussian) or the relative area of the peaks. The equivalent peak resolution value and the classically defined peak resolution value are numerically identical when the separated peaks are Gaussian and have identical areas and SDs. Using our new freeware program, Resolution Analyzer, one can calculate both the classically defined and the equivalent peak resolution values. With the help of this tool, we demonstrate here that the classical peak resolution values mischaracterize the extent of peak overlap even when the peaks are Gaussian but have different areas. We show that under ideal conditions of the separation process, the relative peak overlap value is easily accessible by fitting the overall peak profile as the sum of two Gaussian functions. The applicability of the new approach is demonstrated on real separations.

Inhibitory control and academic achievement - a study of the relationship between Stroop Effect and university students' academic performance.

While previous research has identified executive functions as predictors of academic performance in school children, similar studies conducted among adults show mixed results. One of the reasons given for executive functions having a limited effect on academic achievements in adulthood is that they are usually fully developed by that time. Since these executive functions are at their peak at that age, the individual differences in these as well as their influence on academic performance in adults are harder to trace. The paper describes a study conducted among 107 university students the goal of which was to find out whether there is any relationship between the adult students' inhibitory control values measured with the Stroop Test and their academic achievements. Although the results indicate a weak correlation between the Stroop Effect and the students' academic performance of low statistical significance, which seems to confirm the outcomes of the previous studies focusing on adults, the study reveals an unexpected statistically significant correlation between the students' grade averages and the number of their incorrect color identifications. This phenomenon appears to be worth pursuing in future research since it suggests the existence of another, relatively quickly measurable, variable possibly reflecting other predictors of academic performance in adults such as a degree of their manifested conscientiousness, their ability to concentrate on an assigned, relatively short, one-off task and their attitude to fulfilling this task. The Stroop Test, despite not being originally designed for this purpose, might thus be used as a simple tool suitable for providing information about these variables via the subject's number of color identification errors. Such information can subsequently inform the activities that educators may include in their curricula to foster conscientiousness and concentration in the students lacking these.

Molecular and morphological characterisation of larvae of the genus Diamesa Meigen, 1835 (Diptera: Chironomidae) in Alpine streams (Ötztal Alps, Austria).

Diamesa species (Diptera, Chironomidae) are widely distributed in freshwater ecosystems, and their life cycles are closely linked to environmental variables such as temperature, water quality, and sediment composition. Their sensitivity to environmental changes, particularly in response to pollution and habitat alterations, makes them valuable indicators of ecosystem health. The challenges associated with the morphological identification of larvae invoke the use of DNA barcoding for species determination. The mitochondrial cytochrome oxidase subunit I (COI) gene is regularly used for species identification but faces limitations, such as similar sequences in closely related species. To overcome this, we explored the use of the internal transcribed spacers (ITS) region in addition to COI for Diamesa larvae identification. Therefore, this study employs a combination of molecular markers alongside traditional morphological identification to enhance species discrimination. In total, 129 specimens were analysed, of which 101 were sampled from a glacier-fed stream in Rotmoostal, and the remaining 28 from spring-fed streams in the neighbouring valleys of Königstal and Timmelstal. This study reveals the inadequacy of utilizing single COI or ITS genes for comprehensive species differentiation within the genus Diamesa. However, the combined application of COI and ITS markers significantly enhances species identification resolution, surpassing the limitations faced by traditional taxonomists. Notably, this is evident in cases involving morphologically indistinguishable species, such as Diamesa latitarsis and Diamesa modesta. It highlights the potential of employing a multi-marker approach for more accurate and reliable Diamesa species identification. This method can be a powerful tool for identifying Diamesa species, shedding light on their remarkable adaptations to extreme environments and the impacts of environmental changes on their populations.

Cold mountain stream chironomids (Diptera) of the genus Diamesa indicate both historical and recent climate change.

Chironomids of the genus Diamesa (Meigen, 1835, Diptera: Chironomidae) inhabit cold, oxygen-rich running waters. We have investigated the presence of Diamesa and other freshwater macroinvertebrates at 22 stream sampling sites in 3 European high mountain regions (the Central Pyrenees, the Ötztal Alps, and the Tatra Mountains) to establish suitable temperature conditions for Diamesa dominance. It has been generally accepted that their high abundance was linked to the presence of glaciers; however, we have shown that in the Tatra Mountains, where there are no glaciers, the conditions for the dominance of Diamesa species are created due to permanent snowfields, the geographical orientation of the valley and shading by the surrounding high peaks. The historical connection of Diamesa to glaciers was investigated from the paleolimnological records of subfossil chironomid assemblages from the Bohemian Forest, where glaciers disappeared before or during the Late Glacial period. As expected, water temperature seems to be the main driver of Diamesa distribution, and we determined that the relative abundance of Diamesa species was significantly higher at the sites with a mean July water temperature below 6.5 °C. The Diamesa-dominated stream communities seems to be endangered due to ongoing climate warming and this assumption is supported by our paleolimnological results from the Bohemian Forest lakes, where Diamesa has disappeared due to warming of lake inflows at the beginning of the Holocene. These findings strengthen the former suggestions that some Diamesa species could be used as an indicator for tracking recent environmental changes in vulnerable ecosystems of cold mountain streams.

Applicability and limitations of affinity capillary electrophoresis and vacancy affinity capillary electrophoresis methods for determination of complexation constants.

ACE and vacancy affinity capillary electrophoresis (VACE) are the commonly used methods for determination of complexation constants by CE. The applicability and limitations of these methods were tested experimentally and by means of simulations using our simulation software Simul 5 Complex. It was shown that while the ACE method provides reliable and precise values of complexation parameters, those determined by VACE can be incorrect especially in the case of strong complexation. The effective mobilities of the system peaks in the VACE method, and consequently, the resulting complexation parameters were found to be a function of concentration of the analyte present in the BGE. Development of system peaks in VACE is discussed in the frame of the linear theory of electromigration. Dependence of mobility of system peaks on the composition of the BGE cannot be characterized by a simple analytical expression as in the case of ACE method. Thus, the VACE method fails and the resulting complexation constants might seriously differ from the reality.

What is the quality of pre-dialysis healthcare in the Czech Republic?

AIM: The PREPARE study (PRE-dialysis healthcare in PAtients initiating Renal rEplacement therapy and its consequences) evaluates the quality of pre-dialysis healthcare in patients commencing dialysis treatment in the Czech Republic. METHODS: 48% of Czech dialysis centers participating in this prospective multicenter observational study provided data on all consecutive patients starting renal replacement therapy during 24 weeks. RESULTS: 68% out of 303 patients had nephrological pre-dialysis care lasting >6 months (57% diabetics). Peritoneal dialysis (PD) was chosen by 11.2%. 23.6% of patients were receiving erythropoiesis-stimulating agents while the mean hemoglobin level was 98.3 ±15.6 g/l. 36.1% of patients were taking phosphate binders while serum phosphates reached 1.90 ±0.61 mmol/l. 64.4% of patients had a functional arteriovenous fistula or PD catheter. 91.8% of the patients felt they were well informed about hemodialysis and 51.6% about PD. Physicians reported poor compliance of patients in 15.1% of cases, while the patients evaluated their own compliance as 9.4%. CONCLUSIONS: To conclude: (1) better pre-dialysis care and information are needed; (2) higher awareness on PD might increase its low popularity; (3) particular attention should be paid to diabetics due to their higher morbidity, a lower proportion considered for transplantation and a lower proportion referred to nephrologists by diabetologists, and (4) preemptive transplantation should be considered more often.

Evaluation of a knitted polytetrafluoroethylene mesh placed intraperitoneally in a New Zealand white rabbit model.

BACKGROUND: The intraperitoneal application of surgical mesh remains a controversial issue because of possible complications, especially adhesion and fistula formation. This study aimed to assess the potential of a knitted polytetrafluoroethylene (PTFE) mesh for intraabdominal implantation. METHODS: Twenty-eight 5 × 5 cm samples of knitted macroporous PTFE mesh and light-weight polypropylene mesh (LW-PP) were implanted intraperitoneally in 14 New Zealand white rabbits in a randomized manner and fixed using eight polypropylene stitches. After 90 days, the adhesion formation, adhesion score, shrinkage, strength of fixation to the abdominal wall, and histologic biocompatibility were assessed. RESULTS: No intraoperative or anesthesia-related complications or mesh infection were recorded. The average area covered by adhesions was 4.7 ± 7.2% for the PTFE and 36.4 ± 36.1% for the LW-PP. The median adhesion score was 0 for the PTFE and 8 for the LW-PP. Shrinkage was 36.9 ± 12.9% for the PTFE mesh and 12.6 ± 8.72% for the LW-PP. The mesh-to-abdominal wall fixation strength was almost the same for both materials (PTFE 3.6 ± 1.9 vs. LW-PP 3.6 ± 2.9). The inflammatory cell count was almost the same for the two groups, with no statistically significant difference. The width of the inner granuloma was equal (PTFE 10.5 ± 0.9 vs. LW-PP 11.1 ± 0.9). The outer granuloma was reduced significantly in the PTFE group (PTFE 23.0 ± 2.1 vs. LW-PP 33.6 ± 7.9). One of the animals in the PTFE group died on postoperative day 12 because of ileus. The reason was an adhesion of the small intestine to the polypropylene fixation stitch, which caused small intestine strangulation. CONCLUSIONS: The knitted PTFE mesh induces fewer intraperitoneal adhesions of lower density than the light-weight polypropylene mesh. The strength of the knitted PTFE mesh fixation to the abdominal wall is comparable with that of the light-weight polypropylene mesh, but the shrinkage is greater. The biocompatibility of the knitted PTFE mesh is comparable with that of the light-weight polypropylene implant.

The learning curve of robot-assisted laparoscopic aortofemoral bypass grafting for aortoiliac occlusive disease.

BACKGROUND: Since the end of the 20th century, robot-assisted surgery has been finding its role among other minimally invasive methods. Vascular surgery seems to be another specialty in which the benefits of this technology can be expected. Our objective was to assess the learning curve of robot-assisted laparoscopic aortofemoral bypass grafting for aortoiliac occlusive disease in a group of 40 patients. METHODS: Between May 2006 and January 2010, 40 patients (32 men, 8 women), who were a median age of 58 years (range, 48-75 years), underwent 40 robot-assisted laparoscopic aortofemoral reconstructions. Learning curve estimations were used for anastomosis, clamping, and operative time assessment. For conversion rate evaluation, the cumulative summation (CUSUM) technique was used. Statistical analysis comparing the first and second half of our group, and unilateral-to-bilateral reconstructions were performed. RESULTS: We created 21 aortofemoral and 19 aortobifemoral bypasses. The median proximal anastomosis time was 23 minutes (range, 18-50 minutes), median clamping time was 60 minutes (range, 40-95 minutes), and median operative time was 295 minutes (range, 180-475 minutes). The 30-day mortality rate was 0%, and no graft or wound infection or cardiopulmonary or hepatorenal complications were observed. During the median 18-month follow-up (range, 2-48 months), three early graft occlusions occurred (7%). After reoperations, the secondary patency of reconstructions was 100%. Data showed a typical short learning curve for robotic proximal anastomosis creation with anastomosis and clamping time reduction. The operative time learning curve was flat, confirming the procedure's complexity. There were two conversions to open surgery. CUSUM analysis confirmed that an acceptable conversion rate set at 5% was achieved. Comparing the first and second half of our group, all recorded times showed statistically significant improvements. Differences between unilateral and bilateral reconstructions were not statistically significant. CONCLUSIONS: Our results show that the success rate of robot-assisted laparoscopic aortofemoral bypass grafting is high and the complication rate is low. Anastomosis creation, one of the main difficulties of laparoscopic bypass grafting, has been overcome using the robotic operating system and its learning curve is short. However, the endoscopic dissection of the aortoiliac segment remains the most difficult part of the operation and should be addressed in further development of the method to reduce the operative times. Long-term results and potential benefits of this minimally invasive method have to be verified by randomized controlled clinical trials.

Comparative analysis of selected hydromorphological assessment methods.

The European Water Framework Directive 2000/60/EC aims to achieve a good ecological status of all surface water bodies in Europe. The definition of the ecological status is based on the hydromorphological, hydrochemical, and hydrobiological features of water bodies. Numerous methods are applied for the purpose of hydromorphological status assessment. This study attempts to compare four different methods (EcoRivHab, LAWA Field and Overview Survey, and Rapid Bioassessment Protocol) that were applied at two study areas in the Czech part of the Elbe River Basin. The selected catchments represent areas with different sizes and physical geographic as well as socioeconomic characteristics. All the methods applied showed the capacity to identify the natural and even the completely changed reaches and provided good information on the river physical habitat state. However, they are varied from the viewpoint of the number of parameters, number of monitored zones, time and knowledge demands of the performed assessment.

Metallomics reveals a persisting impact of cadmium on the evolution of metal-selective snail metallothioneins.

The tiny contribution of cadmium (Cd) to the composition of the earth's crust contrasts with its high biological significance, owing mainly to the competition of Cd with the essential zinc (Zn) for suitable metal binding sites in proteins. In this context it was speculated that in several animal lineages, the protein family of metallothioneins (MTs) has evolved to specifically detoxify Cd. Although the multi-functionality and heterometallic composition of MTs in most animal species does not support such an assumption, there are some exceptions to this role, particularly in animal lineages at the roots of animal evolution. In order to substantiate this hypothesis and to further understand MT evolution, we have studied MTs of different snails that exhibit clear Cd-binding preferences in a lineage-specific manner. By applying a metallomics approach including 74 MT sequences from 47 gastropod species, and by combining phylogenomic methods with molecular, biochemical, and spectroscopic techniques, we show that Cd selectivity of snail MTs has resulted from convergent evolution of metal-binding domains that significantly differ in their primary structure. We also demonstrate how their Cd selectivity and specificity has been optimized by the persistent impact of Cd through 430 million years of MT evolution, modifying them upon lineage-specific adaptation of snails to different habitats. Overall, our results support the role of Cd for MT evolution in snails, and provide an interesting example of a vestigial abiotic factor directly driving gene evolution. Finally, we discuss the potential implications of our findings for studies devoted to the understanding of mechanisms leading to metal specificity in proteins, which is important when designing metal-selective peptides.

Distinct pathways for zinc metabolism in the terrestrial slug Arion vulgaris.

In most organisms, the concentration of free Zn2+ is controlled by metallothioneins (MTs). In contrast, no significant proportions of Zn2+ are bound to MTs in the slug, Arion vulgaris. Instead, this species possesses cytoplasmic low-molecular-weight Zn2+ (LMW Zn) binding compound that divert these metal ions into pathways uncoupled from MT metabolism. Zn2+ is accumulated in the midgut gland calcium cells of Arion vulgaris, where they associate with a low-molecular-weight ligand with an apparent molecular mass of ~ 2,000 Da. Mass spectrometry of the semi-purified LMW Zn binding compound combining an electrospray ion source with a differential mobility analyser coupled to a time-of-flight mass spectrometer revealed the presence of four Zn2+-containing ion signals, which arise from disintegration of one higher MW complex resulting in an ion-mobility diameter of 1.62 nm and a molecular mass of 837 Da. We expect that the novel Zn2+ ion storage pathway may be shared by many other gastropods, and particularly species that possess Cd-selective MT isoforms or variants with only very low affinity to Zn2+.