Genetic diversity of Microsporidia in the circulatory system of endemic amphipods from different locations and depths of ancient Lake Baikal

Endemic amphipods (Amphipoda, Crustacea) of the most ancient and large freshwater Lake Baikal (Siberia, Russia) are a highly diverse group comprising >15% of all known species of continental amphipods. The extensive endemic biodiversity of Baikal amphipods provides the unique opportunity to study interactions and possible coevolution of this group and their parasites, such as Microsporidia. In this study, we investigated microsporidian diversity in the circulatory system of 22 endemic species of amphipods inhabiting littoral, sublittoral and deep-water zones in all three basins of Lake Baikal. Using molecular genetic techniques, we found microsporidian DNA in two littoral (Eulimnogammarus verrucosus, Eulimnogammarus cyaneus), two littoral/sublittoral (Pallasea cancellus, Eulimnogammarus marituji) and two sublittoral/deep-water (Acanthogammarus lappaceus longispinus, Acanthogammarus victorii maculosus) endemic species. Twenty sequences of the small subunit ribosomal (SSU) rDNA were obtained from the haemolymph of the six endemic amphipod species sampled from 0–60 m depths at the Southern Lake Baikal’s basin (only the Western shore) and at the Central Baikal. They form clusters with similarity to Enterocytospora, Cucumispora, Dictyocoela, and several unassigned Microsporidia sequences, respectively. Our sequence data show similarity to previously identified microsporidian DNA from inhabitants of both Lake Baikal and other water reservoirs. The results of our study suggest that the genetic diversity of Microsporidia in haemolymph of endemic amphipods from Lake Baikal does not correlate with host species, geographic location or depth factors but is homogeneously diverse

Nonspecific stress response to temperature increase in Gammarus lacustris Sars with respect to oxygen-limited thermal tolerance concept

The previously undescribed dynamics of the heat shock protein HSP70 and subsequent lipid peroxidation products have been assessed alongside lactate dehydrogenase activity for Gammarus lacustris Sars, an amphipod species from the saltwater Lake Shira (Republic of Khakassia). Individuals were exposed to a gradual temperature increase of 1 °C/hour (total exposure duration of 26 hours) starting from the mean annual temperature of their habitat (7 °C) up to 33 °C. A complex of biochemical reactions occurred when saltwater G. lactustris was exposed to the gradual changes in temperature. This was characterized by a decrease in lactate dehydrogenase activity and the launching of lipid peroxidation. The HSP70 level did not change significantly during the entire experiment. In agreement with the concept of oxygen-limited thermal tolerance, an accumulation of the most toxic lipid peroxides (triene conjugates and Schiff bases) in phospholipids occurred at the same time and temperature as the accumulation of lactate. The main criterion overriding the temperature threshold was, therefore, the transition to anaerobiosis, confirmed by the elevated lactate levels as observed in our previous associated study, and by the development of cellular stress, which was expressed by an accumulation of lipid peroxidation products. An earlier hypothesis, based on freshwater individuals of the same species, has been confirmed whereby the increased thermotolerance of G. lacustris from the saltwater lake was caused by differences in energy metabolism and energy supply of nonspecific cellular stress-response mechanisms. With the development of global climate change, these reactions could be advantageous for saltwater G. lacustris. The studied biochemical reactions can be used as biomarkers for the stress status of aquatic organisms when their habitat temperature changes

Temperature Gradient Affects Differentiation of Gene Expression and SNP Allele Frequencies in the Dominant Lake Baikal Zooplankton Species

Local adaptation and phenotypic plasticity are main mechanisms of organisms’ resilience in changing environments. Both are affected by gene flow and are expected to be weak in zooplankton populations inhabiting large continuous water bodies and strongly affected by currents. Lake Baikal, the deepest and one of the coldest lakes on Earth, experienced epilimnion temperature increase during the last 100 years, exposing Baikal’s zooplankton to novel selective pressures. We obtained a partial transcriptome of Epischura baikalensis (Copepoda: Calanoida), the dominant component of Baikal’s zooplankton, and estimated SNP allele frequencies and transcript abundances in samples from regions of Baikal that differ in multiyear average surface temperatures. The strongest signal in both SNP and transcript abundance differentiation is the SW-NE gradient along the 600+ km long axis of the lake, suggesting isolation by distance. SNP differentiation is stronger for nonsynonymous than synonymous SNPs and is paralleled by differential survival during a laboratory exposure to increased temperature, indicating directional selection operating on the temperature gradient. Transcript abundance, generally collinear with the SNP differentiation, shows samples from the warmest, less deep location clustering together with the southernmost samples. Differential expression is more frequent among transcripts orthologous to candidate thermal response genes previously identified in model arthropods, including genes encoding cytoskeleton proteins, heat-shock proteins, proteases, enzymes of central energy metabolism, lipid and antioxidant pathways. We conclude that the pivotal endemic zooplankton species in Lake Baikal exists under temperature-mediated selection and possesses both genetic variation and plasticity to respond to novel temperature-related environmental pressures

A Capital Breeder in a Heterogeneous Environment: Lipid Reserves and RNA:DNA Ratio in Lake Baikal\u27s Endemic Epischura

Lake Baikal (Siberia, Russia) is the oldest, the deepest and the most voluminous lake on Earth. During the last century, Baikal has experienced gradual increases in temperature but remains highly oligotrophic with increases in primary productivity limited to the warmest parts of the lake. Using whole body RNA:DNA ratio as an indicator of metabolic rate, we demonstrate that the key primary consumer in Baikal\u27s plankton, Epischura baikalensis (Copepoda: Calanoida), is a capital breeder that relies on lipid storage to maintain productivity. In individuals from nature, the RNA:DNA ratio correlated with lipid content in samples from cold, low phytoplankton density locations from Baikal\u27s North and Central basins, but not in samples from warmer South basin and Maloe More strait. Lipid reserves, both visually assessed and measured by Nile Red fluorescence, correlated positively with phytoplankton density. In laboratory experiments the RNA:DNA ratio responded to starvation and temperature in non-reproducing, low lipid storage females, but not in individuals with developed ovaries or high lipid storage. This indicates that, unlike many other zooplankton crustaceans, E. baikalensis uses resource storage to support current reproduction, which buffers the dependency of metabolic rate on current feeding conditions. We discuss possible effects of such buffering on E. baikalensis competition with non-endemic, largely income-feeding zooplankton species whose frequency is currently increasing in Baikal pelagia

Nonspecific stress response to temperature increase in Gammarus lacustris Sars with respect to oxygen-limited thermal tolerance concept

The previously undescribed dynamics of the heat shock protein HSP70 and subsequent lipid peroxidation products have been assessed alongside lactate dehydrogenase activity for Gammarus lacustris Sars, an amphipod species from the saltwater Lake Shira (Republic of Khakassia). Individuals were exposed to a gradual temperature increase of 1 ◦C/hour (total exposure duration of 26 hours) starting from the mean annual temperature of their habitat (7 ◦C) up to 33 ◦C. A complex of biochemical reactions occurred when saltwater G. lactustris was exposed to the gradual changes in temperature. This was characterized by a decrease in lactate dehydrogenase activity and the launching of lipid peroxidation. The HSP70 level did not change significantly during the entire experiment. In agreement with the concept of oxygen-limited thermal tolerance, an accumulation of the most toxic lipid peroxides (triene conjugates and Schiff bases) in phospholipids occurred at the same time and temperature as the accumulation of lactate. The main criterion overriding the temperature threshold was, therefore, the transition to anaerobiosis, confirmed by the elevated lactate levels as observed in our previous associated study, and by the development of cellular stress, which was expressed by an accumulation of lipid peroxidation products. An earlier hypothesis, based on freshwater individuals of the same species, has been confirmed whereby the increased thermotolerance ofG. lacustrisfrom the saltwater lake was caused by differences in energy metabolism and energy supply of nonspecific cellular stressresponse mechanisms. With the development of global climate change, these reactions could be advantageous for saltwater G. lacustris. The studied biochemical reactions can be used as biomarkers for the stress status of aquatic organisms when their habitat temperature changes

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

Salinity modulates thermotolerance, energy metabolism and stress response in amphipods Gammarus lacustris

Temperature and salinity are important abiotic factors for aquatic invertebrates. We investigated the influence of different salinity regimes on thermotolerance, energy metabolism and cellular stress defense mechanisms in amphipods Gammarus lacustris Sars from two populations. We exposed amphipods to different thermal scenarios and determined their survival as well as activity of major antioxidant enzymes (peroxidase, catalase, glutathione S-transferase) and parameters of energy metabolism (content of glucose, glycogen, ATP, ADP, AMP and lactate). Amphipods from a freshwater population were more sensitive to the thermal challenge, showing higher mortality during acute and gradual temperature change compared to their counterparts from a saline lake. A more thermotolerant population from a saline lake had high activity of antioxidant enzymes. The energy limitations of the freshwater population (indicated by low baseline glucose levels, downward shift of the critical temperature of aerobic metabolism and inability to maintain steady-state ATP levels during warming) was observed, possibly reflecting a trade-off between the energy demands for osmoregulation under the hypo-osmotic condition of a freshwater environment and protection against temperature stress