Differing temperature dependencies of functional homologs zebrafish Abcb4 and human ABCB1.

The ATP binding cassette (ABC) transporters human ABCB1 and zebrafish (Danio rerio) Abcb4 are functionally homologous multixenobiotic/multidrug (MXR/MDR) efflux transporters that confer the efflux of a broad range of diverse chemical compounds from the cell. As ATPases, the transporters utilize the energy released by ATP cleavage for protein conformation changes and concomitant active transport of substrate compounds. The temperatures, at which human ABCB1 and zebrafish Abcb4 need to function, can substantially differ: Whereas the ambient temperature of human ABCB1, which is that of the human body, is constant, zebrafish Abcb4 has to be active in a wider temperature range as the body temperature of zebrafish can considerably vary, depending on the ambient water temperature (18°C-40°C). Here, we examined the effect of temperature on the ATPase activities of recombinant human ABCB1 and zebrafish Abcb4 generated with the baculovirus expression system. Incubation temperatures for enzyme reactions were set to 37°C and 27°C, corresponding to the human body temperature and the cultivation temperature of zebrafish in our lab, respectively. For stimulation and inhibition of zebrafish Abcb4 and human ABCB1 ATPase activities verapamil and cyclosporin A were added at different concentrations and 50% effect concentrations (EC50) were determined. The different temperatures had a stronger effect on the human ABCB1 than on the zebrafish Abcb4 ATPase: Differences between EC50 values for verapamil at 37°C and 27°C, respectively, were 1.8-fold for human ABCB1 but only 1.2-fold for zebrafish Abcb4. Activation energies (Ea) of basal and verapamil-stimulated ATPases, calculated based on the Arrhenius equation, were 2-fold (basal) and 1.5-fold (verapamil-stimulated) higher for human ABCB1 than for zebrafish Abcb4. The differences between zebrafish Abcb4 and human ABCB1 ATPases in temperature sensitivity and activation energy could be important for the comparison of the functional properties of the two transporter proteins in the context of pharmaco-/toxicokinetics. Related to this, our finding that at equal reaction conditions the zebrafish Abcb4 ATPase activity tended to be generally higher than that of human ABCB1 may also be important, as this may point to a higher substrate compound transport rate of Abcb4.

Preliminary investigation on the potential involvement of an ABC-like gene in Halomicronema metazoicum (Cyanobacteria) tolerance to low seawater pH in an ocean acidification scenario.

Decreasing ocean surface pH, called ocean acidification (OA), is among the major risks for marine ecosystems due to human-driven atmospheric pCO2 increase. Understanding the molecular mechanisms of adaptation enabling marine species to tolerate a lowered seawater pH could support predictions of consequences of future OA scenarios for marine life. This study examined whether the ATP-binding cassette (ABC)-like gene slr2019 confers tolerance to the marine cyanobacterium Halomicronema metazoicum to low seawater pH conditions (7.7, 7.2, 6.5) in short- and long-term exposures (7 and 30 d). Photosynthetic pigment content indicated that the species can tolerate all three lowered-pH conditions. At day 7, slr2019 was up-regulated at pH 7.7 while no changes were observed at lower pH. After 30-d exposure, a significant decrease in slr2019 transcript levels was observed in all low-pH treatments. These first results indicate an effect of low pH on the examined transporter expression in H. metazoicum.

The ABCs of the amphipod P-glycoprotein: Heterologous production of the Abcb1 protein of a model species Eulimnogammarus verrucosus (Amphipoda: Gammaridae) from Lake Baikal.

The multixenobiotic resistance (MXR) mechanism has been demonstrated to be present in a wide range of species, including aquatic organisms. However, amphipods (Crustacea: Malacostraca: Amphipoda), which constitute a large order of arthropods, are extremely poorly studied in this regard. Information on MXR proteins in these animals would be highly relevant, as some amphipods are important models in ecotoxicology due to their roles in many freshwater environments, including the ancient Lake Baikal. In this work, we studied the diversity of ABC transporters in the available transcriptomes of over 60 endemic Baikal amphipods in comparison to other related species. This showed that most classes of ABC transporters are present in all analyzed species and that most Baikal amphipods detectably express no more than one complete ABCB full transporter. We also showed that these sequences were conservative across different species, and their phylogeny was congruent with the species phylogeny. Thus, we chose the abcb1 coding sequence from Eulimnogammarus verrucosus, a widespread species playing an important role in the lake ecosystem, to establish the first heterologous expression system for an amphipod Abcb1/P-glycoprotein based on the Drosophila melanogaster S2 cell line. The resulting stably transfected S2 cell line was expressing the abcb1 of E. verrucosus about 1000 times higher than the homologous fly genes, and the target protein, Abcb1, showed to confer a high MXR-related efflux activity. Our results indicate the suitability of the S2-based expression systems for the study of arthropod ABCB1 homologs.

Cold-adapted amphipod species upon heat stress: Proteomic responses and their correlation with transcriptomic responses.

The cellular heat shock response (HSR) comprises transcriptomic and proteomic reactions to thermal stress. It was here addressed, how the proteomic, together with the transcriptomic HSR, relate to the thermal sensitivities of three cold-adapted but differently thermo-sensitive freshwater amphipod species. The proteomes of thermosensitive Eulimnogammarus verrucosus and thermotolerant Eulimnogammarus cyaneus, both endemic to Lake Baikal, and of thermotolerant Holarctic Gammarus lacustris were investigated upon 24 h exposure to the species-specific 10 % lethal temperatures (LT10). Furthermore, correlations of heat stress induced changes in proteomes (this study) and transcriptomes (previous study with identical experimental design) were examined. Proteomes indicated that the HSR activated processes encompassed (i) proteostasis maintenance, (ii) maintenance of cell adhesion, (iii) oxygen transport, (iv) antioxidant response, and (v) regulation of protein synthesis. Thermo-sensitive E. verrucosus showed the most pronounced proteomic HSR and the lowest correlation of transcriptomic and proteomic HSRs. For proteins related to translation (ribosomal proteins, elongation factors), transcriptomic and proteomic changes were inconsistent: transcripts were downregulated in many cases, with levels of corresponding proteins remaining unchanged. In the Eulimnogammarus species, levels of hemocyanin protein but not transcript were increased upon heat stress, suggesting a HSR also directed to enhance oxygen transport. Thermosensitive E. verrucosus showed the most pronounced relocation of transcription/translation activity to proteostasis maintenance, which may indicate that the general species-specific stability of protein structure could be a fundamental determinant of thermotolerance. By combining transcriptomic and proteomic response data, this study provides a comprehensive picture of the cellular HSR components in the studied amphipods.

Unraveling cellular and molecular mechanisms of acid stress tolerance and resistance in marine species: New frontiers in the study of adaptation to ocean acidification.

Since the industrial revolution, fossil fuel combustion has led to a 30 %-increase of the atmospheric CO2 concentration, also increasing the ocean partial CO2 pressure. The consequent lowered surface seawater pH is termed ocean acidification (OA) and severely affects marine life on a global scale. Cellular and molecular responses of marine species to lowered seawater pH have been studied but information on the mechanisms driving the tolerance of adapted species to comparatively low seawater pH is limited. Such information may be obtained from species inhabiting sites with naturally low water pH that have evolved remarkable abilities to tolerate such conditions. This review gathers information on current knowledge about species naturally facing low water pH conditions and on cellular and molecular adaptive mechanisms enabling the species to survive under, and even benefit from, adverse pH conditions. Evidences derived from case studies on naturally acidified systems and on resistance mechanisms will guide predictions on the consequences of future adverse OA scenarios for marine biodiversity.

Reduced genetic diversity of freshwater amphipods in rivers with increased levels of anthropogenic organic micropollutants.

Anthropogenic chemicals in freshwater environments contribute majorly to ecosystem degradation and biodiversity decline. In particular anthropogenic organic micropollutants (AOM), a diverse group of compounds, including pesticides, pharmaceuticals, and industrial chemicals, can significantly impact freshwater organisms. AOM were found to impact genetic diversity of freshwater species; however, to which degree AOM cause changes in population genetic structure and allelic richness of freshwater macroinvertebrates remains poorly understood. Here, the impact of AOM on genetic diversity of the common amphipod Gammarus pulex (Linnaeus, 1758) (clade E) was investigated on a regional scale. The site-specific AOM levels and their toxic potentials were determined in water and G. pulex tissue sample extracts for 34 sites along six rivers in central Germany impacted by wastewater effluents and agricultural run-off. Population genetic parameters were determined for G. pulex from the sampling sites by genotyping 16 microsatellite loci. Genetic differentiation among G. pulex from the studied rivers was found to be associated with geographic distance between sites and to differences in site-specific concentrations of AOM. The genetic diversity parameters of G. pulex were found to be related to the site-specific AOM levels. Allelic richness was significantly negatively correlated with levels of AOM in G. pulex tissue (p < 0.003) and was reduced by up to 22% at sites with increased levels of AOM, despite a positive relationship of allelic richness and the presence of waste-water effluent. In addition, the inbreeding coefficient of G. pulex from sites with toxic AOM levels was up to 2.5 times higher than that of G. pulex from more pristine sites. These results indicate that AOM levels commonly found in European rivers significantly contribute to changes in the genetic diversity of an ecologically relevant indicator species.

Zebrafish Oatp1d1 Acts as a Cellular Efflux Transporter of the Anionic Herbicide Bromoxynil.

Toxicokinetics (TK) of ionic compounds in the toxico-/pharmacological model zebrafish embryo (Danio rerio) depend on absorption, distribution, metabolism, and elimination (ADME) processes. Previous research indicated involvement of transport proteins in the TK of the anionic pesticide bromoxynil in zebrafish embryos. We here explored the interaction of bromoxynil with the organic anion-transporting polypeptide zebrafish Oatp1d1. Mass spectrometry imaging revealed accumulation of bromoxynil in the gastrointestinal tract of zebrafish embryos, a tissue known to express Oatp1d1. In contrast to the Oatp1d1 reference substrate bromosulfophthalein (BSP), which is actively taken up by transfected HEK293 cells overexpressing zebrafish Oatp1d1, those cells accumulated less bromoxynil than empty vector-transfected control cells. This indicates cellular efflux of bromoxynil by Oatp1d1. This was also seen for diclofenac but not for carbamazepine, examined for comparison. Correspondingly, internal concentrations of bromoxynil and diclofenac in the zebrafish embryo were increased when coexposed with BSP, inhibiting the activities of various transporter proteins, including Oatp1d1. The effect of BSP on accumulation of bromoxynil and diclofenac was enhanced in further advanced embryo stages, indicating increased efflux activity in those stages. An action of Oatp1d1 as an efflux transporter of ionic environmental compounds in zebrafish embryos should be considered in future TK assessments.

Photomotor response data analysis approach to assess chemical neurotoxicity with the zebrafish embryo.

The photomotor response (PMR) of zebrafish embryos, a light pulse-triggered undirected movement, is known to be altered by neuroactive chemicals. Here, we developed an approach for data analysis of the distribution of PMR movement activities along the time axis; differences between treatment and respective controls are expressed by an aggregated value integrating the time-resolved density of the movement parameter as a measure for a chemically elicited PMR effect. Logistic concentration-PMR effect relationships were modeled for neuroactive test compounds with different modes of action (acetylcholinesterase inhibition, activation and inhibition of voltage-gated sodium channels); 50% effect concentrations (EC50) were in the low to medium µM range (EC50 < 10 µM for flucythrinate, esfenvalerate, azinphos-methyl, propoxur; EC50 > 10 µM for tricaine). Modulation of movement activities in different phases of the PMR (i.e., "fingerprint") by neuroactive test compounds varied across concentrations, showing that mode of action-specific PMR fingerprints are also concentration-dependent. Above concentrations causing 10% lethality (LC10; 48 h), 3,4-dichlo­roaniline caused movement inhibition. This substance presumably is not neuroactive; its effect on the PMR therefore is considered a secondary toxic effect. Quantitative morphological examinations of chemically exposed embryos showed that malformations occurred only above PMR effect concentrations, indicating that changes in the PMR were not due to such indirect effects. The PMR assay will provide a useful measure in ecotoxicological risk assessment of neuroactive chemicals with zebrafish embryos and could potentially be used to infer acute fish toxicity levels from PMR effect concen­trations of neurotoxic compounds.

Different ways to play it cool: Transcriptomic analysis sheds light on different activity patterns of three amphipod species under long-term cold exposure.

Species of littoral freshwater environments in regions with continental climate experience pronounced seasonal temperature changes. Coping with long cold winters and hot summers requires specific physiological and behavioural adaptations. Endemic amphipods of Lake Baikal, Eulimnogammarus verrucosus and Eulimnogammarus cyaneus, show high metabolic activity throughout the year; E. verrucosus even reproduces in winter. In contrast, the widespread Holarctic amphipod Gammarus lacustris overwinters in torpor. This study investigated the transcriptomic hallmarks of E. verrucosus, E. cyaneus and G. lacustris exposed to low water temperatures. Amphipods were exposed to 1.5°C and 12°C (corresponding to the mean winter and summer water temperatures, respectively, in the Baikal littoral) for one month. At 1.5°C, G. lacustris showed upregulation of ribosome biogenesis and mRNA processing genes, as well as downregulation of genes related to growth, reproduction and locomotor activity, indicating enhanced energy allocation to somatic maintenance. Our results suggest that the mitogen-activated protein kinase (MAPK) signalling pathway is involved in the preparation for hibernation; downregulation of the actin cytoskeleton pathway genes could relate to the observed low locomotor activity of G. lacustris at 1.5°C. The differences between the transcriptomes of E. verrucosus and E. cyaneus from the 1.5°C and 12°C exposures were considerably smaller than for G. lacustris. In E. verrucosus, cold-exposure triggered reproductive activity was indicated by upregulation of respective genes, whereas in E. cyaneus, genes related to mitochondria functioning were upregulated, indicating cold compensation in this species. Our data elucidate the molecular characteristics behind the different adaptations of amphipod species from the Lake Baikal area to winter conditions.

Low annual temperature likely prevents the Holarctic amphipod Gammarus lacustris from invading Lake Baikal.

Species with effective thermal adaptation mechanisms allowing them to thrive within a wide temperature range can benefit from climatic changes as they can displace highly specialized species. Here, we studied the adaptive capabilities of the Baikal endemic amphipods Eulimnogammarus verrucosus (Gerstfeld, 1858) and Eulimnogammarus cyaneus (Dybowsky, 1874) compared to the potential Holarctic Baikal invader Gammarus lacustris Sars, 1863 at the cellular level including the energy metabolism and the antioxidant system. All species were long-term exposed to a range of temperatures (1.5 °C to mimic winter conditions and the three species-specific preferred temperatures (i.e., 6 °C for E. verrucosus, 12 °C for E. cyaneus and 15 °C for G. lacustris). At 1.5 °C, we found species-specific metabolic alterations (i.e., significantly reduced ATP content and lactate dehydrogenase activity) indicating limitations on the activity level in the Holarctic G. lacustris. Although the two Baikal endemic amphipod species largely differ in thermal tolerance, no such limitations were found at 1.5 °C. However, the cold-stenothermal Baikal endemic E. verrucosus showed changes indicating a higher involvement of anaerobic metabolism at 12 °C and 15 °C, while the metabolic responses of the more eurythermal Baikal endemic E. cyaneus may support aerobic metabolism and an active lifestyle at all exposure temperatures. Rising temperatures in summer may provide a competitive advantage for G. lacustris compared to the Baikal species but the inactive lifestyle in the cold is likely preventing G. lacustris from establishing a stable population in Lake Baikal.

Chemical Pollution Levels in a River Explain Site-Specific Sensitivities to Micropollutants within a Genetically Homogeneous Population of Freshwater Amphipods.

Anthropogenic micropollutants alter chemical and ecological conditions of freshwater ecosystems and impact aquatic species that live along the pollution gradient of a river. Species sensitivity to micropollutants depends on the site-specific exposure; however, it remains unclear to what degree this sensitivity relates to the species' genetic structure. Here, we explored the relationship between the toxic sensitivity and genetic structure of the amphipod species Gammarus pulex (Linnaeus, 1758) along an organic micropollutant gradient in the Holtemme River in central Germany. We determined the river's site-specific micropollutant patterns and analyzed the genetic structure of G. pulex using nuclear and mitochondrial genetic markers. Furthermore, we examined the exposure sensitivities and bioaccumulation of the commonly detected insecticide imidacloprid in G. pulex from different sites. Our results show that throughout the Holtemme River, G. pulex forms a well-connected and homogeneous population with no observable pollution-related differences in the genetic structure. However, G. pulex from polluted sites responded more sensitively to imidacloprid; survival times for half of the amphipods were up to 54% shorter, the percentage of immobile individuals increased up to 65%, and the modeled imidacloprid depuration rate was lower in comparison to amphipods from non-polluted sites. Altogether, these results suggest that the level of sensitivity of G. pulex amphipods to micropollutants in the river depends on the degree of pollution: amphipods may thrive in food-rich but polluted habitats; yet, their sensitivity is increased when chronically exposed to organic micropollutants.

Thermal reaction norms of key metabolic enzymes reflect divergent physiological and behavioral adaptations of closely related amphipod species.

Lake Baikal is inhabited by more than 300 endemic amphipod species, which are narrowly adapted to certain thermal niches due to the high interspecific competition. In contrast, the surrounding freshwater fauna is commonly represented by species with large-scale distribution and high phenotypic thermal plasticity. Here, we investigated the thermal plasticity of the energy metabolism in two closely-related endemic amphipod species from Lake Baikal (Eulimnogammarus verrucosus; stenothermal and Eulimnogammarus cyaneus; eurythermal) and the ubiquitous Holarctic amphipod Gammarus lacustris (eurythermal) by exposure to a summer warming scenario (6-23.6 °C; 0.8 °C d-1). In concert with routine metabolic rates, activities of key metabolic enzymes increased strongly with temperature up to 15 °C in E. verrucosus, whereupon they leveled off (except for lactate dehydrogenase). In contrast, exponential increases were seen in E. cyaneus and G. lacustris throughout the thermal trial (Q10-values: 1.6-3.7). Cytochrome-c-oxidase, lactate dehydrogenase, and 3-hydroxyacyl-CoA dehydrogenase activities were found to be higher in G. lacustris than in E. cyaneus, especially at the highest experimental temperature (23.6 °C). Decreasing gene expression levels revealed some thermal compensation in E. cyaneus but not in G. lacustris. In all species, shifts in enzyme activities favored glycolytic energy generation in the warmth. The congruent temperature-dependencies of enzyme activities and routine metabolism in E. verrucosus indicate a strong feedback-regulation of enzymatic activities by whole organism responses. The species-specific thermal reaction norms reflect the different ecological niches, including the spatial distribution, distinct thermal behavior such as temperature-dependent migration, movement activity, and mating season.

Proteomics reveals sex-specific heat shock response of Baikal amphipod Eulimnogammarus cyaneus.

The ancient Lake Baikal is the largest source of liquid freshwater on Earth and home to a unique fauna. Several hundred mostly cold-adapted endemic amphipod species inhabit Baikal, an ecosystem that is already being influenced by global change. In this study, we characterized the core proteome and heat stress-induced changes in a temperature-tolerant endemic amphipod, Eulimnogammarus cyaneus, using a proteogenomic approach (PRIDE dataset PXD013237) to unravel the molecular mechanisms of the observed adverse effects. As males were previously found to be much more tolerant to thermal stress, we placed special emphasis on differences between the sexes. For both sexes, we observed adaption of energy metabolism, cytoskeleton, lipid, and carbohydrate metabolism upon heat stress. In contrast, significant differences were determined in the molecular chaperone response. Females from the control conditions possessed significantly higher levels of heat shock proteins (HSP70, HSPb1, Hsc70-3), which, in contrast to males, were not further increased in response to heat stress. The inability of females to further increase heat shock protein synthesis in response to temperature stress may be due to sex-specific processes, such as egg production, requiring a large proportion of the available energy. As ovigerous females synthesize generally higher amounts of protein, they also need higher levels of molecular chaperones for the folding of these new proteins. Thus, the higher sensitivity of females to heat shock may be due to the lack of molecular chaperone molecules to counteract the heat-induced protein denaturation.

Chemical effects on dye efflux activity in live zebrafish embryos and on zebrafish Abcb4 ATPase activity.

ATP-binding cassette (ABC) transporter proteins include efflux pumps that confer multixenobiotic resistance to zebrafish embryos, a valuable toxico/pharmacological model. Here, we established an automated microscopy-based rhodamine B dye accumulation assay in which enhanced dye accumulation in live zebrafish embryos indicates inhibition of multixenobiotic efflux transporter activity. Twenty structurally divergent known substrates and/or inhibitors of human ABC transporters and environmentally relevant compounds were examined using this assay and the ATPase activity of recombinant zebrafish Abcb4 as readouts. These two assays confirmed that Abcb4 functions as an efflux transporter in zebrafish, whereas they gave discordant results for some of the tested substances. The dye accumulation assay in zebrafish embryos could be useful to screen environmental pollutants and other chemicals for efflux transporter interaction in a medium-throughput fashion.

Yolk Sac of Zebrafish Embryos as Backpack for Chemicals?

The zebrafish embryo (Danio rerio) has developed into one of the most important nonsentient animal models for the hazard assessments of chemicals, but the processes governing its toxicokinetics (TK) are poorly understood. This study compares the uptake of seven test compounds into the embryonic body and the yolk sac of the zebrafish embryo using TK experiments, a dialysis approach, thermodynamic calculations, and kinetic modeling. Experimental data show that between 95% (4-iodophenol) and 67% (carbamazepine) of the total internal amount in 26 h post fertilization (hpf) embryos and between 80 and 49% in 74 hpf embryos were found in the yolk. Thus, internal concentrations determined for the whole embryo overestimate the internal concentration in the embryonic body: for the compounds of this study, up to a factor of 5. Partition coefficients for the embryonic body and a one-compartment model with diffusive exchange were calculated for the neutral test compounds and agreed reasonably with the experimental data. For prevalently ionic test compounds at exposure pH (bromoxynil, paroxetine), however, the extent and the speed of uptake were low and could not be modeled adequately. A better understanding of the TK of ionizable test compounds is essential to allow assessment of the validity of this organismic test system for ionic test compounds.

Changes of cellular stress response related hsp70 and abcb1 transcript and Hsp70 protein levels in Siberian freshwater amphipods upon exposure to cadmium chloride in the lethal concentration range.

The induction of cellular stress response systems, heat shock protein hsp70/Hsp70 and multixenobiotic transporter abcb1, by cadmium chloride (CdCl2) was explored in amphipod species with different stress adaptation strategies from the Lake Baikal area. Based on the lethal concentrations (LC) of CdCl2, the sensitivities of the different species to CdCl2 were ranked (24 hr LC50 in mg/L CdCl2 (mean/95% confidence interval)): Gammarus lacustris (1.7/1.3-2.4) < Eulimnogammarus cyaneus (2.9/2.1-4.0) < Eulimnogammarus verrucosus (5.7/3.8-8.7) < Eulimnogammarus vittatus (18.1/12.4-26.6). Conjugated dienes, indicating lipid peroxidation, were significantly increased after 24 hr exposures to 5 mg/L CdCl2 only in the more CdCl2-sensitive species G. lacustris and E. cyaneus. Upon treatment with 0.54 to 5.8 mg/L CdCl2 for 1, 6 and 24 hrs, hsp70 transcript levels were generally more increased after the longer exposure times and in the more CdCl2-sensitive species. Relating the CdCl2 exposure concentrations to LCx values revealed that across the species the increases of hsp70 transcript levels were comparatively low (up to 2.6-fold) at CdCl2 concentrations ≤LC50. Relative hsp70 transcript levels were maximally increased in E. cyaneus by 5 mg/L CdCl2 ( = ˆ LC70) at 24 hrs (9.1-fold increase above the respective control). When G. lacustris was exposed to 5 mg/L CdCl2 ( = ˆ LC90) for 24 hrs, the increase in hsp70 was in comparison to E. cyaneus considerably less pronounced (3.0-fold increase in hsp70 levels relative to control). Upon exposure of amphipods to 5 mg/L CdCl2, increases in Hsp70 protein levels compared to untreated controls were highest in E. cyaneus at 1 and 6 hrs (5 mg/L CdCl2 = ˆ LC70) and in E. verrucosus at 24 hrs (5 mg/L CdCl2 = ˆ LC45). Thus, when the fold increases in Hsp70 protein levels in the different amphipod species were related to the respective species-specific LCx values a similar bell-shaped trend as for hsp70 transcript levels was seen across the species. Transcript levels of abcb1 in CdCl2exposed individuals of the different amphipod species varied up to 4.7-fold in relation to the respective controls. In contrast to hsp70/Hsp70, abcb1 transcripts in CdCl2 exposed individuals of the different amphipod species did not indicate similar levels of induction of abcb1 at equal LCx levels across the species. Induction of hsp70 and abcb1 genes and Hsp70 proteins by CdCl2 in the lethal concentration range shows that these cellular responses are rather insensitive to CdCl2 stress in the examined amphipod species. Furthermore, the increase of expression of these cellular defense systems at such high stress levels suggests that induction of these genes is not related to the maintenance of normal metabolism but to mitigation of the effects of severe toxic stress.

Transcriptome-level effects of the model organic pollutant phenanthrene and its solvent acetone in three amphipod species.

Polyaromatic hydrocarbons (PAH) are common pollutants of water ecosystems originating from incineration processes and contamination with mineral oil. Water solubility of PAHs is generally low; for toxicity tests with aquatic organisms, they are therefore usually dissolved in organic solvents. Here we examined the effects of a typical model PAH, phenanthrene, and a solvent, acetone, on amphipods as relevant aquatic invertebrate models. Two of these species, Eulimnogammarus verrucosus and Eulimnogammarus cyaneus, are common endemics of the oligotrophic and pristine Lake Baikal, while one, Gammarus lacustris, is widespread throughout the Holarctic and inhabits smaller and more eutrophic water bodies in the Baikal area. Neither solvent nor phenanthrene caused mortality at the applied concentrations, but both substances affected gene expression in all species. Differential gene expression was more profound in the species from Lake Baikal than in the Holarctic species. Moreover, in one of the Baikal species, E. cyaneus, we found that many known components of the cellular xenobiotic detoxification system reacted to the treatments. Finally, we detected a negative relationship between changes in transcript abundances in response to the solvent and phenanthrene. This mixture effect, weaker than the impact by a single mixture component, needs further exploration.

Isolation and characterization of eleven novel microsatellite markers for fine-scale population genetic analyses of Gammarus pulex (Crustacea: Amphipoda).

The freshwater amphipod species Gammarus pulex (Linnaeus, 1758) is widespread across Europe and Asia and is able to live in a broad range of environmental conditions. Yet, it is of great interest to which degree it is able to tolerate and adapt to the current rapid anthropogenic environmental changes affecting its habitat, such as pollution, changes in river morphology, and invasions of alien gammarid species. Microsatellite primers for genetic population studies with G. pulex have been developed but due to the existence of several genetically different lineages within the species, the application of these primers is not always successful. In order to investigate the impacts of anthropogenic environmental changes on the spatio-genetic patterns of G. pulex lineage E in streams in the Saale river catchment in Germany, we designed eleven novel polymorphic microsatellites for this lineage using a high-throughput sequencing approach. These microsatellites enabled highly specific characterization of three closely related populations. The results show genetically distinct populations reflected by both a principal coordinates analysis and an analysis of molecular variance. Several of the newly designed microsatellite primers also enabled successful cross-amplification of the respective microsatellites in specimens of G. pulex lineage C, while only two microsatellites were amplified successfully and showed polymorphisms for all of the analyzed specimens of G. fossarum Koch, 1836. The microsatellites identified here are suitable for future assessments of micro-evolutionary dynamics of G. pulex from central Germany.

Comparison between transcriptomic responses to short-term stress exposures of a common Holarctic and endemic Lake Baikal amphipods.

BACKGROUND: Lake Baikal is one of the oldest freshwater lakes and has constituted a stable environment for millions of years, in stark contrast to small, transient bodies of water in its immediate vicinity. A highly diverse endemic endemic amphipod fauna is found in one, but not the other habitat. We ask here whether differences in stress response can explain the immiscibility barrier between Lake Baikal and non-Baikal faunas. To this end, we conducted exposure experiments to increased temperature and the toxic heavy metal cadmium as stressors. RESULTS: Here we obtained high-quality de novo transcriptome assemblies, covering mutiple conditions, of three amphipod species, and compared their transcriptomic stress responses. Two of these species, Eulimnogammarus verrucosus and E. cyaneus, are endemic to Lake Baikal, while the Holarctic Gammarus lacustris is a potential invader. CONCLUSIONS: Both Baikal species possess intact stress response systems and respond to elevated temperature with relatively similar changes in their expression profiles. G. lacustris reacts less strongly to the same stressors, possibly because its transcriptome is already perturbed by acclimation conditions.

Indication of ongoing amphipod speciation in Lake Baikal by genetic structures within endemic species.

BACKGROUND: The ancient Lake Baikal is characterized by an outstanding diversity of endemic faunas with more than 350 amphipod species and subspecies. We determined the genetic diversity within the endemic littoral amphipod species Eulimnogammarus verrucosus, E. cyaneus and E. vittatus and investigated whether within those species genetically separate populations occur across Lake Baikal. Gammarus lacustris from water bodies in the Baikal area was examined for comparison. RESULTS: Genetic diversities within a species were determined based on fragments of cytochrome c oxidase I (COI) and for E. verrucosus additionally of 18S rDNA. Highly location-specific haplogroups of E. verrucosus and E. vittatus were found at the southern and western shores of Baikal that are separated by the Angara River outflow; E. verrucosus from the eastern shore formed a further, clearly distinct haplotype cluster possibly confined by the Selenga River and Angarskiy Sor deltas. The genetic diversities within these haplogroups were lower than between the different haplogroups. Intraspecific genetic diversities within E. verrucosus and E. vittatus with 13 and 10%, respectively, were similar to interspecies differences indicating the occurrence of cryptic, morphologically highly similar species; for E. verrucosus this was confirmed with 18S rDNA. The haplotypes of E. cyaneus and G. lacustris specimens were with intraspecific genetic distances of 3 and 2%, respectively, more homogeneous indicating no or only recent disruption of gene flow of E. cyaneus across Baikal and recent colonization of water bodies around Baikal by G. lacustris. CONCLUSIONS: Our finding of separation of subgroups of Baikal endemic amphipods to different degrees points to a species-specific ability of dispersal across areas with adverse conditions and to potential geographical dispersal barriers in Lake Baikal.