Adipose Fin as a Natural "Optical Window" for Implantation of Fluorescent Sensors into Salmonid Fish.

Implantable optical sensors are emerging tools that have the potential to enable constant real-time monitoring of various internal physiological parameters. Such a possibility will open new horizons for health control not only in medicine, but also in animal husbandry, including aquaculture. In this study, we analyze different organs of commonly farmed rainbow trout (Oncorhynchus mykiss) as implantation sites for fluorescent sensors and propose the adipose fin, lacking an endoskeleton, as the optimal choice. The fin is highly translucent due to significantly thinner dermis, which makes the detectable fluorescence of an implanted sensor operating at the visible light range by more than an order of magnitude higher relative to the skin. Compared to the proximal parts of ray fins, the adipose fin provides easy implantation and visualization of the sensor. Finally, we tested fluorescent pH sensors inside the adipose fin and demonstrated the possibility of acquiring their signal with a simple hand-held device and without fish anesthesia. All these features will most likely make the adipose fin the main "window" into the internal physiological processes of salmonid fish with the help of implantable optical sensors.

Durability of Implanted Low-Density Polyacrylamide Hydrogel Used as a Scaffold for Microencapsulated Molecular Probes inside Small Fish.

Implantable sensors based on shaped biocompatible hydrogels are now being extensively developed for various physiological tasks, but they are usually difficult to implant into small animals. In this study, we tested the long-term in vivo functionality of pH-sensitive implants based on amorphous 2.7% polyacrylamide hydrogel with the microencapsulated fluorescent probe SNARF-1. The sensor was easy to manufacture and introduce into the tissues of a small fish Danio rerio, which is the common model object in biomedical research. Histological examination revealed partial degradation of the gel by the 7th day after injection, but it was not the case on the 1st day. Using the hydrogel sensor, we were able to trace the interstitial pH in the fish muscles under normal and hypercapnic conditions for at least two days after the implantation. Thus, despite later immune response, amorphous polyacrylamide is fully suitable for preparing implantable sensors for various mid-term physiological experiments on small fishes. The proposed approach can be further developed to create implantable sensors for animals with similar anatomy.

Histopathological analysis of zebrafish after introduction of non-biodegradable polyelectrolyte microcapsules into the circulatory system.

Polyelectrolyte microcapsules are among the most promising carriers of various sensing substances for their application inside the bloodstream of vertebrates. The long-term effects of biodegradable microcapsules in mammals are relatively well studied, but this is not the case for non-biodegradable microcapsules, which may be even more generally applicable for physiological measurements. In the current study, we introduced non-biodegradable polyelectrolyte microcapsules coated with polyethylene glycol (PMs-PEG) into the circulatory system of zebrafish to assess their long-term effects on fish internal organs with histopathologic analysis. Implantation of PMs-PEG was not associated with the formation of microclots or thrombi in thin capillaries; thus, the applied microcapsules had a low aggregation capacity. The progression of the immune response to the implant depended on the time and the abundance of microparticles in the tissues. We showed that inflammation originated from recognition and internalization of PMs-PEG by phagocytes. These microcapsule-filled immune cells have been found to migrate through the intestinal wall into the lumen, demonstrating a possible mechanism for partial microparticle elimination from fish. The observed tissue immune response to PMs-PEG was local, without a systemic effect on the fish morphology. The most pronounced chronic severe inflammatory reaction was observed near the injection site in renal parenchyma and within the abdominal cavity since PMs-PEG were administered with kidney injection. Blood clots and granulomatosis were noted at the injection site but were not found in the kidneys outside the injection site. Single microcapsules brought by blood into distal organs did not have a noticeable effect on the surrounding tissues. The severity of noted pathologies of the gills was insufficient to affect respiration. No statistically significant alterations in hepatic morphology were revealed after PMs-PEG introduction into fish body. Overall, our data demonstrate that despite they are immunogenic, non-biodegradable PMs-PEG have low potential to cause systemic effects if applied in the minimal amount necessary for detection of fluorescent signal from the microcapsules.

Cellular Immune Response of an Endemic Lake Baikal Amphipod to Indigenous Pseudomonas sp.

Studies of invertebrates have shown that the internal environment of crustaceans is not always sterile in normal conditions, and in many species, it can be populated by microorganisms even in the absence of any visible pathological processes in the body. This observation raises the question of whether genetically modified indigenous hemolymph microorganisms can be used for biotechnological purposes inside the crustacean either as local producers of some compounds or as sensors to physiological parameters. In this study, we tested the ability of the bacteria isolated from the hemolymph of the amphipod Eulimnogammarus verrucosus to hide from the cellular immune response of the host as the most important feature for their potential long-term application in vivo. 16S rDNA amplicon sequencing revealed five common bacterial genera in all analyzed samples of the amphipod hemolymph, among which Pseudomonas is most easily subjected to genome modification and, thus, the most prospective for biotechnological application. Cultivation of Pseudomonas gave us a number of strains undoubtedly derived from the amphipod hemolymph, and one of them (belonging to the Pseudomonas fluorescens group) was chosen for further tests. The primary culture of amphipod hemocytes was used to analyze the immunogenicity of the strain and showed a pronounced reaction of the immune cells to a high amount of the bacteria within six hours. This result indicates that modulation of cellular immune response to metabolically active bacterial cells is not mandatory for the survival and wide distribution of these microorganisms in the hemolymph of numerous amphipod individuals.

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.

Application of PEG-Covered Non-Biodegradable Polyelectrolyte Microcapsules in the Crustacean Circulatory System on the Example of the Amphipod Eulimnogammarus verrucosus.

Layer-by-layer assembled microcapsules are promising carriers for the delivery of various pharmaceutical and sensing substances into specific organs of different animals, but their utility in vivo inside such an important group as crustaceans remains poorly explored. In the current study, we analyzed several significant aspects of the application of fluorescent microcapsules covered by polyethylene glycol (PEG) inside the crustacean circulatory system, using the example of the amphipod Eulimnogammarus verrucosus. In particular, we explored the distribution dynamics of visible microcapsules after injection into the main hemolymph vessel; analyzed the most significant features of E. verrucosus autofluorescence; monitored amphipod mortality and biochemical markers of stress response after microcapsule injection, as well as the healing of the injection wound; and finally, we studied the immune response to the microcapsules. The visibility of microcapsules decreased with time, however, the central hemolymph vessel was confirmed to be the most promising organ for detecting the spectral signal of implanted microencapsulated fluorescent probes. One million injected microcapsules (sufficient for detecting stable fluorescence during the first hours after injection) showed no toxicity for six weeks, but in vitro amphipod immune cells recognize the PEG-coated microcapsules as foreign bodies and try to isolate them by 12 h after contact.

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.

Simple and Effective Administration and Visualization of Microparticles in the Circulatory System of Small Fishes Using Kidney Injection.

The systemic administration of micro-size particles into a living organism can be applied for vasculature visualization, drug and vaccine delivery, implantation of transgenic cells and tiny optical sensors. However, intravenous microinjections into small animals, which are mostly used in biological and veterinary laboratories, are very difficult and require trained personnel. Herein, we demonstrate a robust and efficient method for the introduction of microparticles into the circulatory system of adult zebrafish (Danio rerio) by injection into the fish kidney. To visualize the introduced microparticles in the vasculature, we propose a simple intravital imaging technique in fish gills. In vivo monitoring of the zebrafish blood pH was accomplished using an injected microencapsulated fluorescent probe, SNARF-1, to demonstrate one of the possible applications of the described technique. This article provides a detailed description of the encapsulation of pH-sensitive dye and demonstrates the principles of the quick injection and visualization of the obtained microcapsules for in vivo recording of the fluorescent signal. The proposed method of injection is characterized by a low mortality rate (0-20%) and high efficiency (70-90% success), and it is easy to institute using commonly available equipment. All described procedures can be performed on other small fish species, such as guppies and medaka.

Microencapsulated fluorescent pH probe as implantable sensor for monitoring the physiological state of fish embryos.

In vivo physiological measurement is a major challenge in modern science and technology, as is environment conservation at the global scale. Proper toxicological testing of widely produced mixtures of chemicals is a necessary step in the development of new products, allowing us to minimize the human impact on aquatic ecosystems. However, currently available bioassay-based techniques utilizing small aquatic organisms such as fish embryos for toxicity testing do not allow assessing in time the changes in physiological parameters in the same individual. In this study, we introduce microencapsulated fluorescent probes as a promising tool for in vivo monitoring of internal pH variation in zebrafish embryos. The pH alteration identified under stress conditions demonstrates the applicability of the microencapsulated fluorescent probes for the repeated analysis of the embryo's physiological state. The proposed approach has strong potential to simultaneously measure a range of physiological characteristics using a set of specific fluorescent probes and to finally bring toxicological bioassays and related research fields to a new level of effectiveness and sensitivity.

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.

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.

Thermal Preference Ranges Correlate with Stable Signals of Universal Stress Markers in Lake Baikal Endemic and Holarctic Amphipods.

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.