Effects of salinity on the immune system cells of the tropical sea urchin Echinometra lucunter

https://doi.org/10.1016/j.jembe.2016.09.012Get rights and content

Highlights

  • Effect of the salinity on sea urchin coelomocytes

  • Low salinity increased coelomocytes concentration in the coelomic fluid.

  • Salinity altered ROS levels, ΔΨm and ABC transporters activity in coelomocytes.

  • Salinity did not affect the phagocytic activity.

Abstract

Human activities have caused climate changes and altered the salinity of the oceans. The reduction of the salinity is one of the factors that may limit the distribution and the survival of marine organisms. Coelomocytes are the immune system cells of the echinoderms and have been studied as biomarkers of stress. The aim of the present study was to investigate the effect of the salinity on the immune system cells of the tropical sea urchin Echinometra lucunter. Animals were collected in João Pessoa coast (Brazilian Northeast). Animals or coelomocytes were exposed to different salinities (25, 35, 45) from 4 to 24 h. Phagocytic parameters, production of reactive oxygen species (ROS), mitochondrial activity and ABC transporter activity were analyzed. The phagocytic parameters did not change when animals or cells were exposed to a salinity of 25 or 45 in any time intervals monitored. However, the coelomocytes concentration increased when animals were exposed to the lower salinity. The levels of ROS were higher when cells were incubated at a salinity of 25 but lower when cells were kept at a salinity of 45. It was observed the loss of the mitochondrial inner membrane potential when coelomocytes were incubated at a salinity of 45. The activity of ABC transporters decreased when cells were incubated at the lowest salinity and increased when cells were incubated at the highest salinity tested. The present work shows that the immune system of the tropical sea urchins Ei lucunter tolerates salinity changes from 25 to 45, and suggests two cellular parameters (ROS levels and ABC transporters activity) as potential biomarkers for the monitoring of the impact of environmental salinity changes.

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 (radical dotOH) 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]

References (111)

  • C.Y. Choi et al.

    Molecular characterization and mRNA expression of glutathione peroxidase and glutathione S-transferase during osmotic stress in olive flounder (Paralichthys olivaceus)

    Comp. Biochem. Physiol. A. Mol. Integr. Physiol.

    (2008)
  • S.P.C. Cole et al.

    Transport of glutathione and glutathione conjugates by MRP1

    Trends Pharmacol. Sci.

    (2006)
  • G. Coteur

    Environmental factors influencing the immune responses of the common European starfish (Asterias rubens)

    Fish Shellfish Immunol.

    (2004)
  • M.T. de Faria et al.

    Innate immune response in the sea urchin Echinometra lucunter (Echinodermata)

    J. Invertebr. Pathol.

    (2008)
  • R.P. Ellis et al.

    Immunological function in marine invertebrates: responses to environmental perturbation

    Fish Shellfish Immunol.

    (2011)
  • C. Espinosa-Diez et al.

    Antioxidant responses and cellular adjustments to oxidative stress

    Redox Biol.

    (2015)
  • W.S. Fisher et al.

    Response of European flat oyster (Ostrea edulis) hemocytes to acute salinity and temperature changes

    Aquaculture

    (1987)
  • B. Gagnaire et al.

    Effects of temperature and salinity on haemocyte activities of the Pacific oyster, Crassostrea gigas (Thunberg)

    Fish Shellfish Immunol.

    (2006)
  • Q. Gao et al.

    Mitochondrial DNA protects against salt stress-induced cytochrome c-mediated apoptosis in yeast

    FEBS Lett.

    (2011)
  • P. Gerardi et al.

    Cellular distribution of sea urchin antibacterial activity

    Biol. Cell.

    (1990)
  • S. Gibbons et al.

    Bacterial resistance modifying agents from Lycopus europaeus

    Phytochemistry

    (2003)
  • P.S. Gross et al.

    Echinoderm immunity and the evolution of the complement system

    Dev. Comp. Immunol.

    (1999)
  • T. Hibino et al.

    The immune gene repertoire encoded in the purple sea urchin genome

    Dev. Biol.

    (2006)
  • C.F. Higgins et al.

    Is the multidrug transporter a flippase?

    Trends Biochem. Sci.

    (1992)
  • R.P. Jacoby et al.

    The role of mitochondrial respiration in salinity tolerance

    Trends Plant Sci.

    (2011)
  • C. Jauzein et al.

    Flow cytometric characterization of hemocytes of the sunray venus clam Macrocallista nimbosa and influence of salinity variation

    Fish Shellfish Immunol.

    (2013)
  • P.T. Johnson

    The coelomic elements of sea urchins (Strongylocentrotus) III. In vitro reaction to bacteria

    J. Invertebr. Pathol.

    (1969)
  • M. Kumar et al.

    Biochemical responses of red alga Gracilaria corticata (Gracilariales, Rhodophyta) to salinity induced oxidative stress

    J. Exp. Mar. Biol. Ecol.

    (2010)
  • B. Kurelec et al.

    Distinct glutathione-dependent enzyme activities and a verapamil-sensitive binding of xenobiotics in a fresh-water mussel Anodonta cygnea

    Biochem. Biophys. Res. Commun.

    (1989)
  • B. Kurelec et al.

    Evidence for a multixenobiotic resistance mechanism in the mussel Mytilus galloprovincialis

    Aquat. Toxicol.

    (1991)
  • C. Li et al.

    Expression of antimicrobial peptides in coelomocytes and embryos of the green sea urchin (Strongylocentrotus droebachiensis)

    Dev. Comp. Immunol.

    (2014)
  • Y.-C. Lin et al.

    Modulation of the innate immune system in white shrimp Litopenaeus vannamei following long-term low salinity exposure

    Fish Shellfish Immunol.

    (2012)
  • M.B. Luo et al.

    Salinity-induced oxidative stress and regulation of antioxidant defense system in the marine macroalga Ulva prolifera

    J. Exp. Mar. Biol. Ecol.

    (2011)
  • D.S. Miller

    Regulation of P-glycoprotein and other ABC drug transporters at the blood-brain barrier

    Trends Pharmacol. Sci.

    (2010)
  • C.F.H. Mueller et al.

    Role of the multidrug resistance protein-1 (MRP1) for endothelial progenitor cell function and survival

    J. Mol. Cell. Cardiol.

    (2010)
  • B. Paital et al.

    Effects of temperature on complexes I and II mediated respiration, ROS generation and oxidative stress status in isolated gill mitochondria of the mud crab Scylla serrata

    J. Therm. Biol.

    (2014)
  • M. Perrigault et al.

    Effects of temperature on hard clam (Mercenaria mercenaria) immunity and QPX (Quahog Parasite Unknown) disease development: II. Defense parameters

    J. Invertebr. Pathol.

    (2011)
  • M.P. Russell

    Echinoderm responses to variation in salinity

  • R. Russo et al.

    Transcriptional increase and misexpression of 14-3-3 epsilon in sea urchin embryos exposed to UV-B

    Cell Stress Chaperones

    (2010)
  • M. Schwarzländer et al.

    Monitoring the in vivo redox state of plant mitochondria: effect of respiratory inhibitors, abiotic stress and assessment of recovery from oxidative challenge

    Biochim. Biophys. Acta

    (2009)
  • G. Solaini et al.

    Hypoxia and mitochondrial oxidative metabolism

    Biochim. Biophys. Acta

    (2010)
  • L. Stabili et al.

    Antibacterial activity in the coelomocytes of the sea urchin Paracentrotus lividus

    Comp. Biochem. Physiol. B

    (1996)
  • A. Aderem et al.

    Mechanisms of phagocytosis in macrophages

    Annu. Rev. Immunol.

    (1999)
  • J.D. Allen et al.

    Understanding the effects of low salinity on fertilization success and early development in the sand dollar Echinarachnius parma

    Biol. Bull.

    (2010)
  • J.D. Allen et al.

    Environmental induction of polyembryony in echinoid echinoderms

    Biol. Bull.

    (2015)
  • K. Apel et al.

    Reactive oxygen species: metabolism, oxidative stress, and signal transduction

    Annu. Rev. Plant Biol.

    (2004)
  • J.C. Borges et al.

    Phagocytosis in vitro and in vivo in the Antarctic Sea urchin Sterechinus neumayeri at 0 °C

    Polar Biol.

    (2002)
  • J.C.S. Borges et al.

    Phagocytic amoebocyte sub populations in the perivisceral coelom of the sea urchin Lytechinus variegatus (Lamarck, 1816)

    J. Exp. Zool. A

    (2005)
  • O. Borsani et al.

    Evidence for a role of salicylic acid in the oxidative damage generated by NaCl and osmotic stress in Arabidopsis seedlings

    Plant Physiol.

    (2001)
  • M. Cervello et al.

    Properties of sea urchin coelomocyte agglutinins

    Ital. J. Zool.

    (1996)
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