Description: Eulimnogammarus verrucosus is an amphipod endemic to the unique ecosystem of Lake Baikal and serves as an emerging model in ecotoxicological studies. We report here on a survey sequencing of its genome as a first step to establish sequence resources for this species. From a single lane of paired-end sequencing data, we estimated the genome size as nearly 10 Gb and we obtained an overview of the repeat content. At least two-thirds of the genome are non-unique DNA, and a third of the genomic DNA is composed of just five families of repetitive elements, including low-complexity sequences. Attempts to use off-the-shelf assembly tools failed on the available low-coverage data both before and after removal of highly repetitive components. Using a seed-based approach we nevertheless assembled short contigs covering 33 pre-microRNAs and the homeodomain-containing exon of nine Hox genes. The absence of clear evidence for paralogs implies that a genome duplication did not contribute to the large genome size. We furthermore report the assembly of the mitochondrial genome using a new, guided “crystallization” procedure. The initial results presented here set the stage for a more complete sequencing and analysis of this large genome.

Description: 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.

Description: 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.

Description: Lake Baikal, the world’s most ancient and by volume largest freshwater body on earth, is affected by global change and regional human activities; this concerns littoral regions of the lake in particular. The direct and indirect effects of rising temperatures and pollution on the unique endemic littoral amphipod fauna of Lake Baikal are as yet unresolved. Thus, the aim of this thesis is to provide insights into the physiological processes determining thermal tolerance and toxicant susceptibility in two of the most abundant littoral amphipod species (Eulimnogammarus verrucosus and Eulimnogammarus cyaneus) in comparison with the related gammarid Gammarus lacustris, which is ubiquitously spread in the Holarctic. The three species experience different temperature regimes. G. lacustris experiences the highest thermal fluctuations as it inhabits shallow water habitats, followed by E. cyaneus, which stays in the upper littoral of Lake Baikal throughout the year. By contrast, E. verrucosus migrates to sublittoral areas when temperatures in the upper littoral of Lake Baikal rise in summer. Further, the species differ in body size (body length from the rostrum to the uropods); E. verrucosus (3.5 - 4 cm) is about 3 - 4 times larger than E. cyaneus (ca. 1 cm). G. lacustris (ca. 1 - 1.5 cm) is only slightly larger than E. cyaneus. It inhabits some of Lake Baikal but is not part of the typical littoral amphipod community. Whether global change will promote a widespread invasion of the non-endemic G. lacustris from isolated shallow bays into Lake Baikal is yet unknown. In a comparative framework thermal plasticity of physiological performance parameters was studied on the whole animal, biochemical and molecular level in all three amphipod species under progressive temperature increase (0.8°C d-1; start: 6°C). Toxicant susceptibility was investigated by measuring cadmium (Cd2+) uptake, subcellular cadmium compartmentalization, concentration-mortality relationships and physiological responses to low biologically effective concentrations derived from concentration-mortality relationships. Ventilation rates were limited at lower temperatures in E. verrucosus (10.6°C) than in E. cyaneus (19.1°C) and G. lacustris (21.1°C). These so-called breakpoint temperatures (BPTs) were correlated with migration of E. verrucosus from the upper littoral to deeper and cooler areas. Moreover, there was strong indication that the BPTs of ventilation correspond to the maximal habitat temperature of E. cyaneus and G. lacustris. Thus, within the framework of oxygen- and capacity-limitation of thermal tolerance (OCLTT), it was suggested that the BPTs of ventilation reflect the first level of thermal limitation, i.e. the pejus (“getting worse”) temperatures of the species. Like ventilation, oxygen consumption was constrained at lower temperatures E. verrucosus (15.0°C) than in E. cyaneus (25.2°C) and G. lacustris (23.6°C). Surpassing the BPTs of oxygen consumption led to exponentially increasing mortality. Consequently, the BPTs of oxygen consumption were proposed to correspond to the critical temperatures of the studied species. Temperature-dependent changes in activities of key metabolic enzymes were correlated with those in oxygen consumption rates in all three amphipod species, however, the shapes of curves representing these changes differed between the species. In E. verrucosus, maximal activities of aerobic enzymes in response to changing temperatures followed hyperbolic or peak-shaped curves and, like oxygen consumption rates, decreased at a breakpoints of around 15°C. Only lactate dehydrogenase, which is involved in anaerobic processes increased significantly beyond 15°C. Simultaneously, transcriptional levels of genes coding for enzymes involved in aerobic metabolic processes were down-regulated and genes involved in the response to hypoxia simultaneously up-regulated in E. verrucosus. By contrast, no breakpoint was observed for aerobic enzyme activities of E. cyaneus and G. lacustris. Enzyme acitivities increased exponentially under elevated temperature. While E. cyaneus showed slight thermal compensation through progressively decreasing RNA transcript levels of many enzymes with rising temperature, no thermal compensation was observed for G. lacustris. Consequently, Q10-relationships of enzyme activities at high temperatures were lower in E. cyaneus than in G. lacustris. In contrast to E. verrucosus, smaller-sized E. cyaneus and G. lacustris had faster cadmium uptake rates and thus lower lethal concentrations, likely due to their higher ratio of permeable body surface area to body volume. Subsequent effect-scaled experiments (exposure to species-specific LC1 of CdCl2; G. lacustris had to be excluded due to high cannibalism) revealed that more cadmium was bound to heat stable proteins in E. cyaneus than in E. verrucosus, congruent to its higher cellular stress response capacity. In contrast, exposure to their species-specific LC1, led to similar concentrations of cadmium associated with the metal sensitive fraction (contains cadmium bound to subcellular fractions which includes heat denaturable proteins and cell organelles) in the two species, however, with species-specific physiological responses of the oxygen supply system. Sublethal cadmium concentrations resulted in metabolic depression and reduced ventilation in E. verrucosus but not in E. cyaneus. Furthermore, the combination of cadmium (sublethal concentration at 6°C) and increased temperature was shown to be more deleterious than each single factor alone reflected by elevated mortality in both species. In conclusion, thermal constraints on the oxygen supply system in E. verrucosus, E. cyaneus and G. lacustris may shape the upper temperature limits to the thermal habitats of the species, in line with the theory of an allometry of thermal tolerance and the hypothesis of a systemic to molecular hierarchy of thermal tolerance with the tightest constraints at the highest hierarchical level (whole animal). Concomitant changes at different organizational levels observed for E. verrucosus suggest a tightly regulated system in response to decreasing systemic oxygen availability caused by elevated temperatures. The results presented here underline that both physiological and behavioral responses to changing environmental conditions may determine a species’ success under global change. E. verrucosus has a high behavioral plasticity (mediated by its migration behavior) but low physiological plasticity to cope with rising temperatures. By contrast E. cyaneus has a higher capacity to tolerate current thermal fluctuations in the upper littoral. However, present summer temperatures may already touch the pejus range of E. cyaneus. Consequently, despite the higher thermal tolerance of E. cyaneus it might be more severely affected by future global change than E. verrucosus. Furthermore, despite the fact that lethal concentrations of cadmium were much lower for E. cyaneus, E. verrucosus showed a stronger physiological response (metabolic depression) to concentrations far below lethal ones. Consequently, the extremely sensitive reaction of E. verrucosus to low levels of a chemical stressor underlines that sublethal effects may not necessarily mirror sensitivity rankings based on mortality data. These findings underscore the necessity of water management strategies strictly avoiding chemical contamination of Lake Baikal waters. Rising temperatures likely enhance the deleterious effects of chemical stressors as shown here for cadmium. Whether global change will provide a competitive advantage for G. lacustris in comparison to Baikal endemics and, by extension, promote the widespread invasion of this non-endemic species, could not be elucidated. G. lacustris showed only slightly non-significantly higher heat tolerance than the Baikal endemic E. cyaneus and showed a similar cadmium susceptibility as E. cyaneus. Secondary effects of global change such as eutrophication, which has been found in the littoral of Lake Baikal, are important factors that need to be considered in future studies. Organisms which are sensitive to hypoxic conditions are likely the first to be affected by such changes.

Description: Lake Baikal is a unique ecosystem being the oldest and deepest freshwater lake on earth. Due to stable conditions over millions of years it is suggested that Baikal endemic amphipods are vulnerable to climate change, pollutants and invading species. In this study we investigated the effects of elevated temperature and cadmium levels on the physiological performances of the Baikal endemic amphipods Eulimnogammarus verrucosus and Eulimnogammarus cyaneus and the ubiquitous Palearctic amphipod Gammarus lacustris. This amphipod is regularly introduced into Lake Baikal and a potential invader. We focused on ion and acid/base regulation, oxygen consumption and ventilatory rates as performance parameters. First results showed that magnesium levels in hemolymph decreased during temperature increase (6-30°C, 1°C h-1) in all species investigated while cadmium did not have any effect. Furthermore, Baikal amphipods are not particularly sensitive to cadmium compared to other amphipod species as demonstrated by mortality experiments. The study revealed that E. verrucosus is living under stress conditions when temperatures exceeded 12-14°C while E. cyaneus is able to compensate for the increased oxygen demand up to 27°C (6-30°C, 0.8°C d-1). Given that E. cyaneus and G. lacustris showed similar resting metabolic rates this cannot explain why G. lacustris is not able to establish in Lake Baikal. Differences in the physiological performance under stress conditions between the two studied Baikal species are large. Hence it is supposed to be species-specific to what extent global change will affect Baikal endemics.

Description: Lake Baikal, the world's most voluminous freshwater lake, has experienced unprecedented warming during the last decades. A uniquely diverse amphipod fauna inhabits the littoral zone and can serve as a model system to identify the role of thermal tolerance under climate change. This study aimed to identify sublethal thermal constraints in two of the most abundant endemic Baikal amphipods, Eulimnogammarus verrucosus and Eulimnogammarus cyaneus, and Gammarus lacustris, a ubiquitous gammarid of the Holarctic. As the latter is only found in some shallow isolated bays of the lake, we further addressed the question whether rising temperatures could promote the widespread invasion of this non-endemic species into the littoral zone. Animals were exposed to gradual temperature increases (4 week, 0.8 °C/d; 24 h, 1 °C/h) starting from the reported annual mean temperature of the Baikal littoral (6 °C). Within the framework of oxygen- and capacity-limited thermal tolerance (OCLTT), we used a nonlinear regression approach to determine the points at which the changing temperature-dependence of relevant physiological processes indicates the onset of limitation. Limitations in ventilation representing the first limits of thermal tolerance (pejus (= “getting worse”) temperatures (Tp)) were recorded at 10.6 (95% confidence interval; 9.5, 11.7), 19.1 (17.9, 20.2), and 21.1 (19.8, 22.4) °C in E. verrucosus, E. cyaneus, and G. lacustris, respectively. Field observations revealed that E. verrucosus retreated from the upper littoral to deeper and cooler waters once its Tp was surpassed, identifying Tp as the ecological thermal boundary. Constraints in oxygen consumption at higher than critical temperatures (Tc) led to an exponential increase in mortality in all species. Exposure to short-term warming resulted in higher threshold values, consistent with a time dependence of thermal tolerance. In conclusion, species-specific limits to oxygen supply capacity are likely key in the onset of constraining (beyond pejus) and then life-threatening (beyond critical) conditions. Ecological consequences of these limits are mediated through behavioral plasticity in E. verrucosus. However, similar upper thermal limits in E. cyaneus (endemic, Baikal) and G. lacustris (ubiquitous, Holarctic) indicate that the potential invader G. lacustris would not necessarily benefit from rising temperatures. Secondary effects of increasing temperatures remain to be investigated.

Description: Temperature is the most pervasive abiotic environmental factor for aquatic organisms. Fluctuations in temperature range lead to changes in metabolic performance. Here, we aimed to identify whether surpassing the thermal preference zones is correlated with shifts in universal cellular stress markers of protein integrity, responses to oxidative stress and lactate content, as indicators of anaerobic metabolism. Exposure of the Lake Baikal endemic amphipod species Eulimnogammarus verrucosus (Gerstfeldt, 1858), Ommatogammarus flavus (Dybowski, 1874) and of the Holarctic amphipod Gammarus lacustris Sars 1863 (Amphipoda, Crustacea) to increasing temperatures resulted in elevated heat shock protein 70 (Hsp70) and lactate content, elevated antioxidant enzyme activities (i.e., catalase and peroxidase), and reduced lactate dehydrogenase and glutathione S-transferase activities. Thus, the zone of stability (absence of any significant changes) of the studied molecular and biochemical markers correlated with the behaviorally preferred temperatures. We conclude that the thermal behavioral responses of the studied amphipods are directly related to metabolic processes at the cellular level. Thus, the determined thermal ranges may possibly correspond to the thermal optima. This relationship between species-specific behavioral reactions and stress response metabolism may have significant ecological consequences that result in a thermal zone-specific distribution (i.e., depths, feed spectrum, etc.) of species. As a consequence, by separating species with different temperature preferences, interspecific competition is reduced, which, in turn, increases a species’ Darwinian fitness in its environment.

Description: Eulimnogammarus cyaneus and Eulimnogammarus verrucosus, closely related amphipod species endemic to Lake Baikal, differ with respect to body size (10- to 50-fold lower fresh weights of E. cyaneus) and cellular stress response (CSR) capacity, potentially causing species-related differences in uptake, internal sequestration, and toxic sensitivity to waterborne cadmium (Cd). We found that, compared to E. verrucosus, Cd uptake rates, related to a given exposure concentration, were higher, and lethal concentrations (50%; LC50) were 2.3-fold lower in E. cyaneus (4 weeks exposure; 6 °C). Upon exposures to species-specific subacutely toxic Cd concentrations (nominal LC1; E. cyaneus: 18 nM (2.0 μg L–1); E. verrucosus: 115 nM (12.9 μg L–1); 4 weeks exposure; 6 °C), Cd amounts in metal sensitive tissue fractions (MSF), in relation to fresh weight, were similar in both species (E. cyaneus: 0.25 ± 0.06 μg g–1; E. verrucosus: 0.26 ± 0.07 μg g–1), whereas relative Cd amounts in the biologically detoxified heat stable protein fraction were 35% higher in E. cyaneus. Despite different potencies in detoxifying Cd, body size appears to mainly explain species-related differences in Cd uptake and sensitivities. When exposed to Cd at LC1 over 4 weeks, only E. verrucosus continuously showed 15-36% reduced oxygen consumption rates indicating metabolic depression and pointing to particular sensitivity of E. verrucosus to persisting low-level toxicant pressure.

Description: 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. 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. 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.