Transcriptional response of Saccharomyces cerevisiae to lactic acid enantiomers.

The model yeast, Saccharomyces cerevisiae, is a popular object for both fundamental and applied research, including the development of biosensors and industrial production of pharmaceutical compounds. However, despite multiple studies exploring S. cerevisiae transcriptional response to various substances, this response is unknown for some substances produced in yeast, such as D-lactic acid (DLA). Here, we explore the transcriptional response of the BY4742 strain to a wide range of DLA concentrations (from 0.05 to 45 mM), and compare it to the response to 45 mM L-lactic acid (LLA). We recorded a response to 5 and 45 mM DLA (125 and 113 differentially expressed genes (DEGs), respectively; > 50% shared) and a less pronounced response to 45 mM LLA (63 DEGs; > 30% shared with at least one DLA treatment). Our data did not reveal natural yeast promoters quantitatively sensing DLA but provide the first description of the transcriptome-wide response to DLA and enrich our understanding of the LLA response. Some DLA-activated genes were indeed related to lactate metabolism, as well as iron uptake and cell wall structure. Additional analyses showed that at least some of these genes were activated only by acidic form of DLA but not its salt, revealing the role of pH. The list of LLA-responsive genes was similar to those published previously and also included iron uptake and cell wall genes, as well as genes responding to other weak acids. These data might be instrumental for optimization of lactate production in yeast and yeast co-cultivation with lactic acid bacteria. KEY POINTS: • We present the first dataset on yeast transcriptional response to DLA. • Differential gene expression was correlated with yeast growth inhibition. • The transcriptome response to DLA was richer in comparison to LLA.

Survival, Growth Performance, and Hepatic Antioxidant and Lipid Profiles in Infected Rainbow Trout (Oncorhynchus mykiss) Fed a Diet Supplemented with Dihydroquercetin and Arabinogalactan.

Natural feed supplements have been shown to improve fish viability, health, and growth, and the ability to withstand multiple stressors related to intensive cultivation. We assumed that a dietary mix of plant-origin substances, such as dihydroquercetin, a flavonoid with antioxidative, anti-inflammatory, and antimicrobial properties, and arabinogalactan, a polysaccharide with immunomodulating activity, would promote fish stress resistance and expected it to have a protective effect against infectious diseases. Farmed rainbow trout fish, Oncorhynchus mykiss, received either a standard diet or a diet supplemented with 25 mg/kg of dihydroquercetin and 50 mg/kg of arabinogalactan during a feeding season, from June to November. The fish in the control and experimental groups were sampled twice a month (eight samplings in total) for growth variable estimations and tissue sampling. The hepatic antioxidant status was assessed via the quantification of molecular antioxidants, such as reduced glutathione and alpha-tocopherol rates, as well as the enzyme activity rates of peroxidase, catalase, and glutathione-S-transferase. The lipid and fatty acid compositions of the feed and fish liver were analyzed using thin-layer and high-performance liquid chromatography. The viability, size, and biochemical indices of the fish responded to the growth physiology, environmental variables such as the dissolved oxygen content and water temperature, and sporadic factors. Due to an outbreak of a natural bacterial infection in the fish stock followed by antibiotic treatment, a higher mortality rate was observed in the fish that received a standard diet compared to those fed supplemented feed. In the postinfection period, reduced dietary 18:2n-6 and 18:3n-3 fatty acid assimilation contents were detected in the fish that received the standard diet in contrast to the supplemented diet. By the end of the feeding season, an impaired antioxidant response, including reduced glutathione S-transferase activity and glutathione content, and a shift in the composition of membrane lipids, such as sterols, 18:1n-7 fatty acid, and phospholipids, were also revealed in fish fed the standard diet. Dietary supplementation with plant-origin substances, such as dihydroquercetin and arabinogalactan, decreases lethality in fish stocks, presumably though the stimulation of natural resistance in farmed fish, thereby increasing the economic efficacy during fish production. From the sustainable aquaculture perspective, natural additives also diminish the anthropogenic transformation of aquaculture-bearing water bodies and their ecosystems.

Antibacterial Activity of Rainbow Trout Plasma: In Vitro Assays and Proteomic Analysis.

The objective of this study was to investigate the bactericidal activity of blood plasma from cultured rainbow trout obtained from two different fish farms. Plasma from trout naturally infected with the bacterial pathogen Flavobacterium psychrophilum was found to inhibit the growth of Aeromonas hydrophila in vitro. Incubation of A. hydrophila in bacteriostatic trout plasma resulted in agglutination and growth retardation, without causing massive damage to the cell membrane. The proteome of the plasma with high antimicrobial activity revealed an abundance of high-density apolipoproteins, some isoforms of immunoglobulins, complement components C1q and C4, coagulation factors, lectins, periostin, and hemoglobin. Analysis of trout proteins retained on A. hydrophila cells revealed the presence of fish immunoglobulins, lectins, and complement components on bacteria whose growth was inhibited, although the native membrane attack complex of immunised trout plasma did not assemble effectively, resulting in a weak bactericidal effect. Furthermore, this study examined the bacterial response to trout plasma and suggested that the protein synthesis pathway was the target of antimicrobial proteins from fish blood. Taken together, these findings illustrate the advantages of the affinity approach for understanding the role of plasma proteins in host defence against pathogens.

Tissue Reaction to Low-Density Polyacrylamide Gel as a Carrier for Microimplants in the Adipose Fin of Rainbow Trout.

The implantation of optical sensors is a promising method for monitoring physiological parameters of organisms in vivo. For this, suitable hydrogels are required that can provide a biocompatible interface with the organism's tissues. Amorphous hydrogel is advantageous for administration in animal organs due to its ease of injection compared to resilient analogs. In this study, we investigated the applicability of a semi-liquid 2.5% polyacrylamide hydrogel (PAAH) as a scaffold for fluorescent polyelectrolyte microcapsules (PMs) in rainbow trout. The hydrogel was injected subcutaneously into the adipose fin, which is a small, highly translucent fold of skin in salmonids that is convenient for implanting optical sensors. Using histological methods, we compared tissue organization and in vivo stability of the applied hydrogel at the injection site after administration of uncoated PMs or PMs coated with 2.5% PAAH (PMs-PAAH) for a period of 3 to 14 days. Our results showed that the introduction of PMs into the gel did not have a masking effect, as they were recognized, engulfed, and carried away by phagocytes from the injection site. However, both PMs and PMs-PAAH were found to provoke chronic inflammation at the injection site, although according to cytokine expression in the fish spleen, the irritating effect was local and did not affect the systemic immunity of the fish. Therefore, our study suggests low applicability of 2.5% polyacrylamide as a scaffold for injectable sensors within a timeframe of days.

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.

SSU rRNA sequencing data for bacterial communities associated with Vibrio-infected rainbow trout (Oncorhynchus mykiss).

Vibrio anguillarum infection in aquacultured trout, besides its own harmful effects, can also make the fish more susceptible to other infections, both by swamping the host's immune system and by creating the skin lesions that serve as a direct gateway for opportunistic bacteria. Tissue samples were taken from the intestines, spleen and skin lesions of cage-grown rainbow trout suffering from natural Vibrio infections of varying severity. In addition, a water sample was taken to document the planktonic bacteria inhabiting the fish farm at the time of the study. Total DNA was isolated from these samples and used to produce v3-v4 SSU rRNA amplicons, which were then sequenced on Illumina MiSeq (2 × 300 bp) and assembled into 97% identity OTUs. This dataset allows analysis of bacteria co-colonizing the skin and intestines of infected fish along with the pathogenic Vibrio anguillarum, as well as provides information on the spread of bacteria between various bodily compartments of trout.

Physiological and Biochemical Characteristics of Rainbow Trout with Severe, Moderate and Asymptomatic Course of Vibrio anguillarum Infection.

This article describes the clinical manifestation of natural Vibrio anguillarum infection in rainbow trout (Oncorhynchus mykiss) during an outbreak on a fish farm. (i) Using an integrated approach, we characterized the pathogenesis of vibriosis from the morphological, hematological, and biochemical points of view. The molecular mechanisms associated with the host immune response were investigated using mass spectrometric analysis of trout plasma proteins. (ii) According to the severity of infection (the extent of tissue damage, the level of expression of pro-inflammatory genes, and changes in the leukocyte profile) three fish populations were identified among infected trout: fish with severe lesions (SL), fish with the moderate infectious process (IP) and asymptomatic fish (AS). (iii) Lymphopenia, granulocytosis, and splenomegaly were strong trends during the progression of infection and informative indicators of severe manifestation of disease, associated with hemorrhagic shock, metabolic acidosis, and massive tissue damage. (iv) As expected, pro-inflammatory interleukins, complement components, acute phase proteins, and antimicrobial peptides were implicated in the acute pathogenesis. Systemic coagulopathy was accompanied by increased antithrombotic reactions. (v) Reconstruction of metabolic pathways also revealed a high energy requirement for the immune response in severely affected fish. (vi) An unexpected result was a small difference between fish with moderate symptoms and fish with no or minor external signs of pathology (putatively resistant to infection). Increased production of antiproteases and enhanced blood coagulation cascade were observed in healthier fish, which may underlie the mechanisms of a controlled, non-self-damaging immune response to infection. (vii) Depending on the progression of the disease and the presence of the pathogen, a stepwise or linear change in the abundance of some plasma proteins was revealed. These proteins could be proposed as molecular markers for diagnosing the health and immune status of trout when cultured in fish farms.

Zone of Interaction Between the Parasite and the Host: Protein Profile of the Body Cavity Fluid of Gasterosteus aculeatus L. Infected with the Cestode Schistocephalus solidus (Muller, 1776).

PURPOSE: During infection, the host and the parasite "communicate" with each other through various molecules, including proteins. The aim of this study was to describe the excretory-secretory proteins from the helminth Schistocephalus solidus and its intermediate host, the three-spined stickleback Gasterosteus aculeatus L., which are likely to be involved in interactions between them. METHODS: Combined samples of washes from the G. aculeatus sticklebacks cavity infected with the S. solidus, and washes from the parasite surface were used as experimental samples, while washes from the uninfected fish body cavity were used as control. The obtained samples were analyzed using mass-spectrometry nLC-MS/MS. RESULTS: As a result of mass-spectrometry analysis 215 proteins were identified. Comparative quantitative analysis revealed significant differences in LFQ intensity between experimental and control samples for 20 stickleback proteins. In the experimental samples, we found an increase in the content of serpins, plasminogen, angiotensin 1-10, complement component C9, and a decrease in the content of triosephosphate isomerase, creatine kinase, fructose-biphosphate aldolase, superoxide dismutase, peroxidoxin-1, homocysteine-binding and fatty acid-binding proteins, compared to uninfected fish samples. In the experimental group washes, 30 S. solidus proteins were found, including malate dehydrogenase, annexin family proteins, serpins, peptidyl-prolyl cis-trans isomerase and fatty acid-binding protein. CONCLUSIONS: Thus, the protein composition of washes from the helminth S. solidus surface and the body cavity of infected and uninfected stickleback G. aculeatus were studied. As a result, it was shown that various components of the immune defense system predominated in the washes of infected fish and helminths.

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.

Comparative Analysis of Proteins of Functionally Different Body Parts of the Fish Parasites Triaenophorus nodulosus and Triaenophorus crassus.

PURPOSE: Studies of proteins expressed in the morphological structures of the parasite are necessary for elucidating the biological functions of unknown proteins and understanding the molecular basis of parasitism. The research aim was to investigate the spatial distribution of major proteins in scolex, immature and gravid proglottids of Triaenophorus nodulosus and Triaenophorus crassus. METHODS: Protein extracts of worm body parts were analyzed using two-dimensional difference gel electrophoresis (DIGE) and mass spectrometry. RESULTS: Comparison of the protein repertoire of the adult worm and the encysted plerocercoid revealed differences between the worm body parts, life stages and parasite species. The content of proteins associated with the cytoskeleton and musculature (actin, myosin regulatory light chain, and tropomyosin 2) decreased with distance from the scolex. Mature proglottids were rich in transforming growth factor-beta-induced protein, propionyl-CoA carboxylase, glutamate dehydrogenase and beta-tubulin. Interspecific variation in T. nodulosus and T. crassus was found in the content of the myosin, paramyosin, the major vault protein and an uncharacterized secreted protein TRINITY_DN24645. Differential expression of TRINITY_DN24645, paramyosin and tropomyosin 2 was found between plerocercoids and adult worms. CONCLUSION: The present study provides the first characteristics of the spatial distribution of the major proteins of T. crassus and T. nodulosus. Comparison of the protein composition of plerocercoids and adult parasites indicates a significant similarity in the proteomic organization of Triaenophorus sp. in the second intermediate and final hosts. The gradual change in the morphological organization of tapeworms in the longitudinal direction coincided with the expression of some structural and metabolic proteins.

Temperature-induced reorganisation of Schistocephalus solidus (Cestoda) proteome during the transition to the warm-blooded host.

The protein composition of the cestode Schistocephalus solidus was measured in an experiment simulating the trophic transmission of the parasite from a cold-blooded to a warm-blooded host. The first hour of host colonisation was studied in a model experiment, in which sticklebacks Gasterosteus aculeatus infected with S. solidus were heated at 40°C for 1 h. As a result, a decrease in the content of one tegument protein was detected in the plerocercoids of S. solidus. Sexual maturation of the parasites was initiated in an experiment where S. solidus larvae were taken from fish and cultured in vitro at 40°C for 48 h. Temperature-independent changes in the parasite proteome were investigated by incubating plerocercoids at 22°C for 48 h in culture medium. Analysis of the proteome allowed us to distinguish the temperature-induced genes of S. solidus, as well as to specify the molecular markers of the plerocercoid and adult worms. The main conclusion of the study is that the key enzymes of long-term metabolic changes (glycogen consumption, protein production, etc.) in parasites during colonisation of a warm-blooded host are induced by temperature.

Dataset on body weight and length of rainbow trout, Oncorhynchus mykiss, fed with dihydroquercetin, arabinogalactan or a mixture of both in an aquaria experiment.

The use of natural dietary supplements in aquaculture has received a great deal of attention in recent years. This article provides data describing body weight and length of rainbow trout juveniles fed with natural dietary supplements dihydroquercetin, arabinogalactan or a mixture of both in an aquaria experiment. Before feeding trial, rainbow trout were tagged to identify individuals. Fish grown in tanks were fed one of four diets in duplicate: a basal diet without any supplements (control diet) or a basal diet supplemented with dihydroquercetin (experimental diet 1), arabinogalactan (experimental diet 2) or a mixture of both (experimental diet 3). Our dataset could be used to evaluate the effect of dihydroquercetin, arabinogalactan or a mixture of both on the growth performance of cultivated rainbow trout.

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.

The effect of Triaenophorus nodulosus (Cestoda: Bothriocephalidea) infection on some biochemical parameters of the liver of Perca fluviatilis.

Natural infection of 2 to 6-year-old perch with the cestode parasites Triaenophorus nodulosus was shown to have minor effects on the studied components of the antioxidant defense system, nucleic acids degradation, and carbohydrate metabolism enzymes in the liver of the fish. The level of infection of 1-4 parasite larvae per fish observed in wild population of perch was shown to be moderate in terms of its effect on the health of the host fish. The activity of hepatic enzymes ß-galactosidase, ß-glucosidase, cathepsin D, and glutathione S-transferase showed different responses in infected males and females, which indicates different potential resistance of fish to the stress exposure between genders.

Correction to: The effect of Triaenophorus nodulosus (Cestoda: Bothriocephalidea) infection on some biochemical parameters of the liver of Perca fluviatilis.

[This corrects the article DOI: 10.1007/s12639-019-01128-0.].

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.

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.