Effects of salinity on the immune system cells of the tropical sea urchin Echinometra lucunter
Introduction
Deuterostome invertebrates have developed a variety of immune responses against foreign pathogens and molecules (Gross et al., 1999). The genomic sequencing of the sea urchin Strongylocentrotus purpuratus revealed that these animals have a robust immune system which is comprised by a vast repertoire of genes involved in the innate recognition of pathogen molecules (Sodergren et al., 2006). The main mechanisms of immune defense against infectious agents used by the most of the invertebrates are: a) synthesis and secretion of proteins/molecules with recognition, neutralizing or lytic activity, which participate in nodule formation; b) encapsulation, phagocytosis of foreign particles and cell lysis (Cervello et al., 1996, Gerardi et al., 1990, Li et al., 2014, Loker et al., 2004, Majeske et al., 2013, Stabili et al., 1996, Tahseen, 2009).
In echinoderms, the immune response is divided into humoral (mediated by molecules present in the body fluids) and cellular (cell mediated). The cells responsible for the innate immunity are known as coelomocytes. The coelomocytes are found in the coelomic perivisceral cavity, vascular water system, circulatory system, connective tissue and the tissues of various organs (Tahseen, 2009); and consist of four subpopulations: phagocytes; vibratile cells; red spherule cells and colorless spherule cells (Johnson, 1969). The phagocytes are involved in cell migration and phagocytosis, and represent the major subpopulation of coelomocytes. Phagocytosis is a mechanism in which immune cells migrate to the infected site, recognize, ingest and destroy the foreign particle (inert or alive) and is the main immune defense mechanism of the marine invertebrates (Ellis et al., 2011). Several phagocytic receptors, such as Toll-like receptors and scavengers receptors, are responsible for the phagocytic process in the specialized cells (Aderem and Underhill, 1999). It is estimated that about 4 to 5% of the genes in the genome of S. purpuratus are directly involved with the immune system (Hibino et al., 2006).
The production of reactive oxygen species (ROS) is a cellular process associated with phagocytosis and the response to the stress (Buggé et al., 2007, Coteur, 2004, Forman and Torres, 2002, Lesser, 2006). The reactive oxygen species are produced during phagocytosis to destroy the internalized particles (Forman and Torres, 2002). The process begins on the cell or phagosome membranes by activating NADPH oxidase, followed by strong oxygen consumption, in a mechanism known as oxidative burst. This process leads to the reduction of molecular oxygen to superoxide anion (O2−); which can be spontaneously or enzymatically (superoxide dismutase) converted into hydrogen peroxide (H2O2). Other reactive radicals, such as hydroxyl radical (OH) or singlet oxygen (1O2), are also produced (Dupré-crochet et al., 2013). Furthermore, ROS are also produced by the mitochondrial electron transport chain under physiological or stress conditions (Banh et al., 2016, Bhat et al., 2015, Figueira et al., 2013, Kandola et al., 2015, Orrenius et al., 2007). When an imbalance between ROS production and ROS scavenger occurs, the cell enters in a state named oxidative stress (Costantini and Verhulst, 2009). Several works have been using the ROS levels of sea urchins immune system cells as biomarker of stress from different sources, such as: UV radiation, acute heat shock, pH reduction or heavy metals exposure (Coteur et al., 2005, Lu and Wu, 2005, Matranga et al., 2000).
Another important defense mechanism present in deuterostome invertebrates which acts against physical and chemical stressors is the activity of the ABC transporters (Bonaventura et al., 2011, Bonaventura et al., 2005, Dean, 2001, Miller, 2010, Russo et al., 2010). The ABC transporters constitute a large group of integral membrane proteins that promote the active transport of a substrate across the membrane. In eukaryotes, ABC transporters are found in the plasma membrane, but also in the membranes that constitute the endomembrane system (Babakhanian et al., 2007, Burke and Ardehali, 2007, Gibbons et al., 2003, Higgins and Gottesman, 1992, ter Beek et al., 2014, Zutz et al., 2009). The ABC proteins are widely distributed - from microorganisms to human - and their structures are highly conserved (Dean et al., 2001). These transporters were firstly associated with the multidrug resistance phenomenon in cancer cells (Gottesman et al., 2002, Rumjanek et al., 2001). Recently, ABC transporters have been linked to cellular detoxification and associated with the protection of marine organisms against xenobiotics (multixenobiotic resistance, also known as MXR) (Ferreira et al., 2014, Kurelec and Pivčević, 1991, Kurelec and Pivčević, 1989, Kurelec, 1992). The sequenced genome of the sea urchins S. purpuratus revealed a wide range of genes encoding ABC transporters (Sodergren et al., 2006). The expression of ABC transporters is regulated in response to xenobiotics, stress and diseases (Bonaventura et al., 2005, de Araujo Leite et al., 2014, Felix and Barrand, 2002, Miller, 2010). Recently, it has been reported that ABC transporters may also play an important role in immune system processes, such as phagocytosis and cell migration (Hinz and Tampé, 2012, Seyffer and Tampé, 2014, van de Ven et al., 2009).
Marine ecosystems play a key role in the ecology of the planet. Human activities have caused climate changes which have altered the hydrological cycle of the planet, including: increase in the incidence of ultraviolet radiation, rise of the sea surface temperature, acceleration in the global rainfall, changes in the rate of evaporation and changes in the salinity of the oceans (Haerter et al., 2010, Semenov et al., 2012, Talley et al., 2002, Trenberth, 1998, Williamson et al., 2014). The reduction of the salinity is one of the main factors that limits the survival and the distribution of marine species (Kaiser, 2011, Li et al., 2013, Russell, 2013, Tomanek et al., 2012). Curry et al. (2003) reported that global warming and changes in the hydrological cycle have altered the distribution of water in the oceans all over the world (Curry et al., 2003). These changes have effects on the physiology and survival of several organisms (Allen and Pechenik, 2010, Carballeira et al., 2011, Choi et al., 2013, Kumar et al., 2010, Luo and Liu, 2011). Some works have reported that fertilization rate, embryo cleavage and polyspermy occurrence - in echinoderms - are sensitive to salinity variations (Allen and Pechenik, 2010, Allen et al., 2015, Carballeira et al., 2011). In spite of being an osmoconformer, echinoderms are stenohaline animals and do not tolerate large variations in the salinity of the environment (Freire et al., 2011). For the sea urchins, fluctuations in environmental salinity may be reflected in the coelomic fluid and impact the physiology of the coelomocytes. However, some studies have shown that Echinoidea species are able to tolerate moderate salinity changes (Drouin et al., 1985, Stickle and Denoux, 1976, Wolff, 1968).
Benthic marine organisms can act as excellent biosensors on the monitoring of the effects of stress on the marine ecosystem. Several studies have demonstrated that sea urchin immune cells respond to environmental stressors, such as: temperature (Borges et al., 2002, Pinsino et al., 2008), UV radiation (Matranga et al., 2006) and pollutants (Pinsino et al., 2008). It has also been described that salinity can affect marine invertebrate immune system, reducing the immune response to foreign agents (Fisher et al., 1987). The tropical sea urchin Echinometra lucunter inhabits intertidal areas and is subject to environmental changes such as temperature and salinity (Lima et al., 2009). The investigation of the status of sea urchin immune system cells under different salinity conditions may contribute to the understanding of the effects of environmental changes in the marine ecosystem. Adding to this, the sea urchin reproductive process plays a relevant role in the marine ecosystem as an important component of the marine food chain. So, the aim of the present study was to investigate the effect of salinity on immune system cells of the tropical sea urchin E. lucunter, contributing to the knowledge about the effects of climate change on the physiology of marine invertebrates.
Section snippets
Drugs
Calcein-AM (C/AM), carbonyl cyanide m-chlorophenyl hydrazone (CCCP), 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA), MK571, reversin 205, hydrogen peroxide (H2O2) and DiOC6(3) (3,3′-Dihexyloxacarbocyanine,iodide) were purchased from Sigma-Aldrich (St. Louis USA). Fluorescent latex beads were purchased from Polysciences, Inc. (Pennsylvania, USA) and NaCl, KCl, CaCl22H2O, MgCl26H2O, MgSO47H2O and NaHCO3 were purchased from VETEC Química Fina (Rio de Janeiro, Brazil).
Animals capture and maintenance
Adult sea urchins E.
Effect of salinity on the phagocytosis capacity at different time spans - in vivo assays
Firstly, it was investigated the phagocytic capacity of coelomocytes from animals exposed to different salinities. The analysis was performed immediately before the exposure to different salinities (0 h) and 6 h and 24 h after the exposure to the lower or higher salinities. The basal phagocytosis (time 0 h) ranged from 51.03 ± 2.69% to 61.73 ± 3.41% according to the treatment group (Fig. 1A). The phagocytic capacity of the sea urchin coelomocytes did not change when animals were kept at different
Discussion
In the present study, it was investigated the effect of salinity on sea urchin immune system cells. Sea urchins - whole animals - or coelomocytes were exposed to ambient (35), low (25) or high salinity (45) in order to assess their sensitivity to the abiotic factor. The present results showed that changes in the salinity did not affect the phagocytic capacity, but ROS production and ABC transporters activity were sensitive to different salinities.
Firstly, it was investigated the effect of
Author contribution
Luis Fernando Marques-Santos defined the research theme, designed the protocols, performed all data analyses, analyzed the results and wrote the manuscript. Thais Mangeon Honorato designed the protocols, performed all data analyses, analyzed the results and wrote the manuscript. Patricia Mirella da Silva co-designed the protocols, co-discussed all results and wrote the manuscript. Thaís Bezerra Mangeon Honorato and Raianna Boni de Vasconcelos carried out the experiments.
Acknowledgements
This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (Project number: 479206/2012-0). Thaís Bezerra Mangeon Honorato and Raianna Boni are recipients of a graduate fellowship from the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). [SS]
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