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.

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.

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.

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.

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