Metabolic Tolerance to Atmospheric Pressure of Two Freshwater Endemic Amphipods Mostly Inhabiting the Deep-Water Zone of the Ancient Lake Baikal.

Lake Baikal is the only freshwater reservoir inhabited by deep-water fauna, which originated mostly from shallow-water ancestors. Ommatogammarus flavus and O. albinus are endemic scavenger amphipods (Amphipoda, Crustacea) dwelling in wide depth ranges of the lake covering over 1300 m. O. flavus had been previously collected close to the surface, while O. albinus has never been found above the depth of 47 m. Since O. albinus is a promising model species for various research, here we tested whether O. albinus is less metabolically adapted to atmospheric pressure than O. flavus. We analyzed a number of energy-related traits (contents of glucose, glycogen and adenylates, as well as lactate dehydrogenase activity) and oxidative stress markers (activities of antioxidant enzymes and levels of lipid peroxidation products) after sampling from different depths and after both species' acclimation to atmospheric pressure. The analyses were repeated in two independent sampling campaigns. We found no consistent signs of metabolic disturbances or oxidative stress in both species right after lifting. Despite O. flavus surviving slightly better in laboratory conditions, during long-term acclimation, both species showed comparable reactions without critical changes. Thus, the obtained data favor using O. albinus along with O. flavus for physiological research under laboratory conditions.

The level of putative carotenoid-binding proteins determines the body color in two species of endemic Lake Baikal amphipods.

Color is an essential clue for intra- and interspecies communication, playing a role in selection and speciation. Coloration can be based on nanostructures and pigments; carotenoids and carotenoproteins are among the most widespread pigments in animals. Over 350 species and subspecies of amphipods (Crustacea: Amphipoda) endemic to Lake Baikal exhibit an impressive variability of colors and coloration patterns, including intraspecific color morphs. However, the mechanisms forming this diversity are underexplored, as while the carotenoid composition of several transparent, green, and red species was investigated, there have been no reports on the corresponding carotenoid-binding proteins. In this work, we analyze the coloration of two brightly colored Baikal amphipods characterized by intraspecific color variability, Eulimnogammarus cyaneus and E. vittatus. We showed that the color of either species is defined by the level of putative carotenoid-binding proteins similar to the pheromone/odorant-binding protein family, as the concentration of these putative crustacyanin analogs was higher in blue or teal-colored animals than in the orange- or yellow-colored ones. At the same time, the color did not depend on the total carotenoid content, as it was similar between animals of contrasting color morphs. By exploring the diversity of these sequences within a larger phylogeny of invertebrate crustacyanins, we show that amphipods lack orthologs of the well-studied crustacyanins A and C, even though they possess some crustacyanin-like sequences. The analysis of expression levels in E. cyaneus showed that the transcripts encoding crustacyanin analogs had much higher expression than the crustacyanin-like sequences, suggesting that the former indeed contribute to the color of these brightly colored animals. The crustacyanin analogs seem to act in a similar way to the well-studied crustacyanins in body color formation, but the details of their action are still to be revealed.

Distribution of PEG-coated hollow polyelectrolyte microcapsules after introduction into the circulatory system and muscles of zebrafish.

The use of polyelectrolyte multilayer microcapsules as carriers for fluorescent molecular probes is a prospective technique for monitoring the physiological characteristics of animal vasculature and interstitial environment in vivo Polyelectrolyte microcapsules have many features that favor their use as implantable carriers of optical sensors, but little information is available on their interactions with complex living tissues, distribution or residence time following different routes of administration in the body of vertebrates. Using the common fish model, the zebrafish Danio rerio, we studied in vivo the distribution of non-biodegradable microcapsules covered with polyethylene glycol (PEG) over time in the adults and evaluated potential side effects of their delivery into the fish bloodstream and muscles. Fluorescent microcapsules administered into the bloodstream and interstitially (in concentrations that were sufficient for visualization and spectral signal recording) both showed negligible acute toxicity to the fishes during three weeks of observation. The distribution pattern of microcapsules delivered into the bloodstream was stable for at least one week, with microcapsules prevalent in capillaries-rich organs. However, after intramuscular injection, the phagocytosis of the microcapsules by immune cells was manifested, indicating considerable immunogenicity of the microcapsules despite PEG coverage. The long-term negative effects of chronic inflammation were also investigated in fish muscles by histological analysis.

Parallel in vivo monitoring of pH in gill capillaries and muscles of fishes using microencapsulated biomarkers.

Tracking physiological parameters in different organs within the same organism simultaneously and in real time can provide an outstanding representation of the organism's physiological status. The state-of-the-art technique of using encapsulated fluorescent molecular probes (microencapsulated biomarkers) is a unique tool that can serve as a platform for the development of new methods to obtain in vivo physiological measurements and is applicable to a broad range of organisms. Here, we describe a novel technique to monitor the pH of blood inside the gill capillaries and interstitial fluid of muscles by using microencapsulated biomarkers in a zebrafish model. The functionality of the proposed technique is shown by the identification of acidification under anesthesia-induced coma and after death. The pH in muscles reacts to hypoxia faster than that in the gill bloodstream, which makes both parameters applicable as markers of either local or bodily reactions.

Intersexual differences of heat shock response between two amphipods (Eulimnogammarus verrucosus and Eulimnogammarus cyaneus) in Lake Baikal.

Acute temperature fluctuations are common in surface waters, and aquatic organisms may manifest physiological responses to punctuated temperature spikes long before behavioral responses. Ectotherms, especially cryophilic stenotherms such as those endemic to Lake Baikal (Siberia), may demonstrate specialized physiological responses to acute temperature increases because their proteomes have evolved to function most efficiently at lower temperatures (e.g., <10 °C). Therefore, our study questioned the nature and degree of variation in physiological response to acute thermal stress in two congenerous, endemic Baikal amphipod species, Eulimnogammarus verrucosus and Eulimnogammarus cyaneus. We hypothesized that because interspecific and intersexual thermosensitivity varies significantly among ectotherms, there would be divergent intersexual and interspecific strategies to withstand acute thermal stress, manifested in different protein compositions and concentrations. We exposed individuals to the species' respective LT50 for one hour followed by a three-hour recovery period. We then performed 1D-PAGE, Western blotting, 2D-PAGE, and Mass Spectrometry techniques and assessed relative intersexual and interspecific changes in proteomic composition and heat shock protein 70 level. Our results demonstrate that females tend to be more sensitive to an acute thermal stimulus than males, most likely because females allocate significant energy to reproduction and less to heat shock response, evidenced by females' significantly lower LT50time. Lower level of Hsp70 was found in females of the thermosensitive E. verrucosus compared to males of this species. No intersexual differences were found in Hsp70 level in thermotolerant E. cyaneus. Higher levels of hemocyanin subunits and arginine kinase were found in E. cyaneus females after heat shock and recovery compared to males, which was not found for E. verrucosus, suggesting interspecific mechanisms for E. cyaneus's higher thermotolerance. These differing responses between species and sexes of Baikal amphipods may reflect more general strategies for maintaining homeostatic conditions during acute thermal stress. As mean surface water temperatures increase worldwide, the net efficiency and efficacy of these strategies could give rise to long term changes in physiology, behavior, and interactions with other species, potentially precipitating population and community level alterations.

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.

Remote in vivo stress assessment of aquatic animals with microencapsulated biomarkers for environmental monitoring.

Remote in vivo scanning of physiological parameters is a major trend in the development of new tools for the fields of medicine and animal physiology. For this purpose, a variety of implantable optical micro- and nanosensors have been designed for potential medical applications. At the same time, the important area of environmental sciences has been neglected in the development of techniques for remote physiological measurements. In the field of environmental monitoring and related research, there is a constant demand for new effective and quick techniques for the stress assessment of aquatic animals, and the development of proper methods for remote physiological measurements in vivo may significantly increase the precision and throughput of analyses in this field. In the present study, we apply pH-sensitive microencapsulated biomarkers to remotely monitor the pH of haemolymph in vivo in endemic amphipods from Lake Baikal, and we compare the suitability of this technique for stress assessment with that of common biochemical methods. For the first time, we demonstrate the possibility of remotely detecting a change in a physiological parameter in an aquatic organism under ecologically relevant stressful conditions and show the applicability of techniques using microencapsulated biomarkers for remote physiological measurements in environmental monitoring.