© 2019. Published by The Company of Biologists Ltd | Journal of Experimental Biology (2019) 222, jeb198556. doi:10.1242/jeb.198556 RESEARCH ARTICLE Injury affects coelomic fluid proteome of the common starfish, Asterias rubens Sergey V. Shabelnikov1,*, Danila E. Bobkov2, Natalia S. Sharlaimova2 and Olga A. Petukhova2 ABSTRACT and exceeding 40% in sea urchins (Giese, 1966). Cellular elements 5– 6 −1 Echinoderms, possessing outstanding regenerative capabilities, found in CF at concentrations of 10 10 cells ml are collectively provide a unique model system for the study of response to injury. referred to as coelomocytes (Chia and Xing, 1996). Their However, little is known about the proteomic composition of coelomic morphology in starfish is well described (Kanungo, 1984; fluid, an important biofluid circulating throughout the animal’s body Sharlaimova et al., 2014). Coelomocytes are not only mediators of and reflecting the overall biological status of the organism. In this innate immune responses (Smith et al., 2010), but are also actively study, we used LC-MALDI tandem mass spectrometry to characterize involved in the early repair phase of starfish arm regeneration (Ben the proteome of the cell-free coelomic fluid of the starfish Asterias Khadra et al., 2017; Ferrario et al., 2018), providing wound closure ‘ ’ rubens and to follow the changes occurring in response to puncture and hemostatic activity. The number of circulating coelomocytes in wound and blood loss. In total, 91 proteins were identified, of which starfish rapidly increases in the first hours after arm tip amputation 61 were extracellular soluble and 16 were bound to the plasma (Pinsino et al., 2007) or an immune challenge (Coteur et al., 2002; membrane. The most represented functional terms were ‘pattern Holm et al., 2008), indicating the importance of cellular components recognition receptor activity’ and ‘peptidase inhibitor activity’. A series in the early post-traumatic period. of candidate proteins involved in early response to injury was Previously, two injury models have been established in Asterias revealed. Ependymin, β-microseminoprotein, serum amyloid A and rubens (Kozlova et al., 2006; Sharlaimova et al., 2014) to study the avidin-like proteins, which are known to be involved in intestinal dynamics of the CF cell population during the early regeneration regeneration in the sea cucumber, were also identified as injury- period: puncture wound (PW) and blood loss (BL). The PW model is responsive proteins. Our results expand the list of proteins potentially characterized by making a single puncture into the body wall, leading involved in defense and regeneration in echinoderms and to slight bleeding, whereas BL involves arm tip amputation, demonstrate dramatic effects of injury on the coelomic fluid proteome. excessive bleeding and artificial washing of the coelomic cavity, effectively reducing the CF cell pool by up to 90% (Sharlaimova KEY WORDS: Echinoderms, Biofluid, Injury response, Wound, et al., 2014). These injuries are clearly different in their consequences: HPLC, LC-MALDI PW induces a rapid and transient increase in the number of circulating coelomocytes in the few hours after injury with its cellular INTRODUCTION composition remaining unchanged (Gorshkov et al., 2009; Kozlova Extracellular body fluids, such as blood plasma in vertebrates et al., 2006), whereas BL is followed by changes in the cellular and hemolymph and coelomic fluid (CF) in invertebrates, serve as a compositions of both CF and coelomic epithelium, upregulation of transport system for metabolites, nutrients, signaling and defense protein synthesis in coelomocytes, and migration of small, poorly molecules in virtually all animals. Extracellular body fluids contain differentiated cells from the coelomic epithelium into the CF soluble factors that are constitutively secreted as well as those that (Kozlova et al., 2006; Sharlaimova et al., 2014). Involvement of are released under specific physiological conditions from different small, poorly differentiated cells in the renewal of CF cell populations sources throughout the entire body, providing an opportunity to after BL injury has been proposed (Sharlaimova et al., 2014). develop an overall profile of the biological status of the organism. Echinoderms, having an outstanding capability to regenerate body Various soluble factors are directly or indirectly involved in parts and even complete individuals from a fragment following self- different physiological processes, such as wound healing, induced or traumatic amputation processes, represent valuable models inflammation, tissue remodeling and cell migration. These soluble in regeneration research (Ben Khadra et al., 2017; Candia-Carnevali factors will leak into the extracellular body fluids, serving as et al., 2009; Dolmatov, 1999). Recent high-throughput studies of biomarkers of their corresponding processes. regeneration in sea cucumbers (Dolmatov et al., 2018; Zhang et al., Echinoderms have a well-developed perivisceral coelom 2017) have enabled discovery of new players involved in this process; (Lawrence, 1987) in which CF circulates through the whole body, unfortunately, nothing is known about the protein composition of the bathing internal tissues and organs. The volume of CF constitutes a CF in these animals. Previous proteomic studies of the echinoderm CF significant fraction of the whole body mass, reaching 20% in starfish have generally focused on immune-challenge- and age-related changes in sea urchins (Bodnar, 2013; Dheilly et al., 2011; Dheilly et al., 2012; Dheilly et al., 2013). These studies analyzed whole CF 1Laboratory of Regulation of Gene Expression, Institute of Cytology, Russian without thorough removal of coelomocytes, which apparently Academy of Sciences, 194064 St Petersburg, Russia. 2Department of Cell Cultures, Institute of Cytology, Russian Academy of Sciences, 194064 St Petersburg, Russia. hampered identification of scarce extracellular components in favor of highly abundant intracellular proteins. Although several proteomic *Author for correspondence ([email protected]) studies have been performed on starfish analyzing mucous secretions S.V.S., 0000-0002-5693-5310 (Hennebert et al., 2015), nervous system regeneration (Franco et al., 2014) and coelomocytes (Franco et al., 2011), none have addressed Received 19 December 2018; Accepted 11 February 2019 the protein composition of CF or its injury-related changes. Journal of Experimental Biology 1 RESEARCH ARTICLE Journal of Experimental Biology (2019) 222, jeb198556. doi:10.1242/jeb.198556 Here, we present a proteomic analysis of cell-free CF samples Animals and experimental injury from A. rubens. This echinoderm provides a suitable model Experiments were performed at the Biological Station of the for proteomic analysis of the CF for the following reasons. First, Zoological Institute, Russian Academy of Sciences, on Cape regenerating specimens of A. rubens are frequently found in the Kartesh (Kandalaksha Bay, White Sea) in September 2016. Adult wild, suggesting the existence of well-developed healing individuals of the common starfish, Asterias rubens Linnaeus 1758, mechanisms. Second, A. rubens has well-developed perivisceral were collected off Fettakh Island (66°20′05″ N, E33°39′07″ E). coelom in terms of both volume and size, permitting multiple Asterias rubens is not a protected or endangered species. Animals rounds of collection of CF. Third, there are three A. rubens were kept in cages at a depth of 5–6 m throughout the experimental transcriptomic datasets available (Hennebert et al., 2015; period. We also included a 3-day acclimation and starvation period Reich et al., 2015; Semmens et al., 2016), enabling the creation prior to experimentation in order to minimize possible perturbations of a species-specific protein database. Forth, A. rubens is a of CF caused by the animal feeding and capture procedure. The common and easily obtained species of starfish in the White Sea starfish used had a radius ranging from 5 to 10 cm, measured from the in Russia. largest arm to the center of the oral disc. Two types of experimental Essentially, the main question we ask is: if an animal with high traumatic treatment were used: puncture wound (PW) and blood loss regenerative capacity and well-developed coelom is subjected to (BL). The experimental workflow is shown in Fig. 1. The PW (n=12 injury, can we identify the soluble proteins involved and are these individuals, mean radius=8.3 cm) was inflicted by puncturing the related to regeneration? To address this question, we examined CF aboral surface of the arm with a scalpel. The puncture resulted in an in the course of the early repair phase in two well-established injury incision of approximately 10 mm and minor bleeding. The BL injury models. The overall aims of this study were to (1) provide an (n=8 individuals, mean radius=5.4 cm) was performed as follows: the overview of the starfish cell-free CF proteome and (2) describe the arm tip was cut off, and the CF was drained off as completely as impact of injury on CF composition and reveal the injury-related possible. Then, the coelomic cavity was washed with four 4 ml soluble factors involved. injections of 0.22 µm-filtered seawater into each arm tip, and the injected seawater was drained off to remove residual CF and MATERIALS AND METHODS coelomocytes. The injured animals were returned to their cages. Ethics statement Steps were taken to ensure that animals did not suffer unnecessarily Experimental