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

Abstract

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.

Figure 1

(a) General scheme of the preparation of MBMs based on the pH-sensitive fluorescent dye SNARF-1 (here, only 3 layers of positively charged polymer, 3 layers of negatively charged polymer and a final coating of biocompatible polymer are depicted). (b) Image of the prepared MBMs recorded using an epifluorescent microscope. (c) Calibration curve of the pH-sensitive MBMs at different pHs (mean ± s.d. is depicted). (d) E. verrucosus, with the main body segments, central haemolymph vessel and point of injection of MBMs indicated. (e) System used for amphipod immobilisation under the epifluorescent microscope.

Figure 2

(a) Variation in the autofluorescence spectrum of the dorsal part of E. verrucosus among different individuals. (b) Spectra of protonated SNARF-1 (microencapsulated, at pH 2), deprotonated SNARF-1 (microencapsulated, at pH 8.9) and autofluorescence of E. verrucosus used as standards for spectral decomposition with multiple linear regression. The dashed lines show the range used for spectral decomposition.

Figure 3. In vivo monitoring of the haemolymph pH of E. verrucosus under different conditions: right after the injection of MBMs and 6 h after the injection; under elevated CO₂ level; and exposure without aeration.

The blue colour indicates acclimation conditions, whereas orange indicates stress exposure. *Statistically significant differences from the control with a p-value < 0.05.

Figure 4. Dynamics of lactate concentration under different conditions: right after the injection of MBMs and 6 h after the injection; under elevated CO₂ level; and exposure without aeration.

The blue colour indicates the acclimation conditions, whereas orange indicates stress exposure. *Statistically significant differences with a p-value < 0.05.