**1. Introduction**

Various implantable nano- and micro-scaled structures have grea<sup>t</sup> potential for becoming the foundation of novel technologies for monitoring and manipulating the state of diverse organisms. Such structures are, for example, polyelectrolyte microcapsules produced by the layer-by-layer (LbL) adsorption technique [1,2]. This technique is based on the deposition of some polymers onto various colloidal microparticles [3–5], including porous structures, such as calcium carbonate in the form of vaterite that can be preloaded with certain substances and further dissolved to obtain soft microcapsules with the substances inside [6,7]. These microcapsules can be easily prepared, using either biodegradable

or non-biodegradable polymers to control their stability inside an organism for different applications [3]. Strongly charged poly(allylamine hydrochloride) (PAH) and poly(sodium 4-styrenesulfonate) (PSS) are among the most popular non-biodegradable polyelectrolytes used as sequential building blocks for microcapsule shells [8–10]. These two polymers form bilayers with a structure close to lamellar and can be used to prepare shells of given thickness [9].

For increasing the overall biocompatibility of the microcapsules, a polyethylene glycol (PEG) coating was suggested [11]. PEG can be attached to polyelectrolyte microcapsules in the form of a graft copolymer, with the charged main polymer sticking to the microcapsule surface electrostatically, while backbones of PEG obduce the microcapsule and reduce their aggregation and friction, which simplifies their distribution in the circulatory system [12,13], as well as decreasing protein adsorption to the surface, which can lower or at least postpone the immune response [11,13].

Crustaceans are an important source of aquatic food protein and popular research objects in ecophysiology [14,15]. However, possibilities for the application of microcapsules as an implantable tool for monitoring and adjusting the physiological status of crustaceans in vivo remain poorly explored. Previously, we applied the PEG-covered polyelectrolyte microcapsules to deliver the pH-sensitive fluorescent dye SNARF-1 into the main hemolymph vessel of the amphipod *Eulimnogammarus verrucosus* (Figure 1) and monitor pH changes in vivo [16]. The same approach is perspective for measuring other parameters, such as the concentration of different ions and metabolites [17], directly in the circulatory system of various crustaceans with translucent exoskeletons. The microcapsules used were composed of non-biodegradable PAH and PSS, to increase their stability and prolong the possibility of sensing.

**Figure 1.** A photo of the amphipod *Eulimnogammarus verrucosus* and schematic designation of all body parts where the microcapsules were observed (colored by green) after injection into the circulatory system. The red dashed line indicates the approximate position of the digestive system. The microcapsules contained fluorescein (in the form of FITC-albumin) and were monitored in the green channel.

In the present study, we analyze various nuances arising from the application of microencapsulated fluorescent dyes in the crustacean circulatory system, using the example of the same amphipod *E. verrucosus* (Figure 1), endemic to Lake Baikal [18]. In particular, we monitor the visibility of the PEG-coated PAH/PSS microcapsules after injection into the main hemolymph vessel and analyze the autofluorescence of *E. verrucosus* to highlight the most promising body parts for the application of microencapsulated molecular probes. Then, we assess the survival and stress response of the amphipods after injection of the microcapsules. Finally, we study the wound healing after injection and the amphipod immune response to the microcapsules.

#### **2. Materials and Methods**
