SHORT COMMUNICATION ISJ 11: 192-196, 2014 ISSN 1824-307X SHORT COMMUNICATION First report of phenoloxidase and peroxidase activities in two intertidal sea anemone species of Argentina AV Fernández Gimenez, NS Haran, NA Pereira, FH Acuña Instituto de Investigaciones Marinas y Costeras (CONICET-Universidad Nacional de Mar del Plata), Funes 3350, 7600 Mar del Plata, Argentina Accepted June 6, 2014 Abstract The presence of immune responses within sea anemone species has received little attention, in comparison with coral species, so we decided to investigate the phenoloxidase and peroxidase activities in ectoderm, endoderm and tentacles of actiniarians Aulactinia marplatensis and Bunodosoma zamponii, the most common species in intertidal zone of Mar del Plata, Argentina. Enzyme activities were detected in all tissues evaluated with some differences among tissues and species. Phenoloxidase and peroxidase activities are associated with the mechanisms of innate immunity in invertebrates, and the high production of phenoloxidase observed in B. zamponii would provide a continual level of resistance to infection and this species to be less susceptible to stress and disease, compared to A. marplatensis. This study, represents the first step toward specific immune information about the mentioned sea anemone species of Argentina, and thus permits prediction of the potential effects of environmental factors on immune response. Key Words: disease; immunity; peroxidase; phenoloxidase; sea anemone; stress; tissues   Introduction Numerous studies have demonstrated that environmental factor variations such as temperature, salinity, oxygen, nutrients and contaminants can strongly affect immune parameters in invertebrates. In this context, immunomarkers have been proposed to be sensitive tools in eco-immunology studies to detect signs of impaired animal health (Matozzo et al., 2013). Palmer et al. (2010) concluded that immunological parameters, such as phenoloxidase activity, provide good indicator of coral immunity and underpin linkages between the susceptibility of corals to disease. Immunity refers to the ability of an organism to resist infection with the nonspecific and immediate innate immune pathways providing the first line of internal defense. A key component of invertebrate innate immunity is the presence and activation of the melanin-synthesis pathway in response to invasion by foreign organisms or physical injury (Rinkevich, 2004). Melanin pathway activity, as ___________________________________________________________________________ Corresponding author: Analia Fernández Gimenez Instituto de Investigaciones Marinas y Costeras Facultad de Ciencias Exactas y Naturales Universidad Nacional de Mar del Plata Funes 3350. 7600, Mar del Plata, Argentina E mail: fgimenez@mdp.edu.ar indicated by levels of the activating enzyme phenoloxidase, has been documented in scleractinian corals, gorgonians and true soft corals from the Caribbean and Indo-Pacific (Palmer et al., 2008, 2011; Mydlarz et al., 2009; Mydlarz and Palmer, 2011). Furthermore, melanin is a redox- active pigment and therefore has the potential not only to be cytotoxic and kill pathogens, but also to scavenge oxygen radicals that may be harmful to the host. Oxygen radical scavengers and enzymatic antioxidants are important during infection, as host responses frequently induce oxidative stress conditions (Palmer et al., 2011). Several studies have linked cnidarian peroxidase activity with antioxidant potential (Hawkridge et al., 2000; Olano and Bigger, 2000) and oxidation of fatty acid hydroperoxides (Koljak et al., 1997). Mydlarz and Harvell (2007) argued that enzymatically driven resistance measures, such as peroxidase activity, are important in the early responses of the sea fan Gorgonia ventalina to a fungal pathogen Aspergillus sydowii. As immunity determines, the ability of an organism to resist and eliminate infection and to recover from injury, it can be used as a predictor of compromised health susceptibility. The immune defenses increase fitness by promoting survival, through disease resistance and the maintenance of tissue integrity. The study of marine invertebrate 192 mailto:fgimenez@mdp.edu.ar ecological immunity is advancing very quickly and critical signaling pathways and cytotoxic responses are being elucidated. The presence and relative activities of phenoloxidase and antioxidants, such as, peroxidase were investigated in several coral species, mainly scleractinian (Mydlarz and Harvell, 2006). The presence of immune responses within sea anemone species have received little or virtually no attention, but their biology suggests that disease- resisting defenses would be adaptive. Hutton and Smith (1996) investigated the antimicrobial defenses of Actinia equina and Hawkridge et al. (2000) determined the subcellular distribution of antioxidant enzymes in the temperate sea anemone Anemonia viridis. According to the scarce knowledge on this topic related with sea anemones we decided to investigate the phenoloxidase and peroxidase activities in ectoderm, endoderm and tentacles of actiniarians Aulactinia marplatensis and Bunodosoma zamponii. These species are the most common species in the rocky intertidal zone of Mar del Plata (38º 05’ S-57º 32’W). They are found mainly attached to hard quartzitic substrate and the taxonomical status of both species was studied by Acuña et al. (2007) and Braga Gomes et al. (2012), respectively. Many aspects of their biology and ecology were also studied, like those related with reproduction (Zamponi and Excoffon, 1986; Excoffon and Zamponi, 1991, 1997), population ecology (Acuña and Zamponi, 1995a, 1996a, 1998), feeding (Acuña and Zamponi, 1995b; 1996b; Acuña, 1997; Acuña et al., 1999), as well as particular topics like analyses of the cnidae (Acuña and Zamponi, 1997) and mycosporine-like amino acid content (Arbeloa et al., 2010). However other aspects, like immunological, remain unknown. This study, represents the first record toward specific immune information about the mentioned sea anemone species of Argentina, and thus permits prediction of the potential effects of environmental factors on immune response. Materials and Methods Sample collection specimens of the sea anemones Aulactinia marplatensis and Bunodosoma zamponii were obtained from the intertidal zone of the rocky area with a quartzitic substrate in Punta Cantera, Mar del Plata (38° 05’S and 57° 38’W). The individuals, all around the same size (30 mm in basal diameter), were caught in December 2012 in the same area for both species during the low tide, but covered by water. The organisms, n = 10 for each species, were maintained at room temperature in an aquarium with decanted and aerated sea water and they were sampled one day after collection. For both anemone species, tissue of epidermis, endodermis (gastrodermis) and tentacles, were removed and homogenized with a 50 mM phosphate buffer at pH 7.8 on ice. Samples were then centrifuged for 5 min at 4,000 rpm and 4 °C, avoiding the mucus layer and the supernatant (protein extract) was carefully removed and stored at -20 °C. Soluble protein in protein extract was measured by the method described by Bradford (1976), using chicken egg white albumin as the standard. Phenoloxidase and Peroxidase activities were determined according to Palmer et al. (2011). Phenoloxidase was assay using 50 µl of protein extract; 100 µl of phosphate buffer (50 mM pH 7.8) and 50 µl double distilled water pyrogen free were incubated for 20 min at room temperature, then 50 µl L-DOPA (3 mg ml-1) (Aldrich, 333786) was added and after 10 min 350 µl of cacodylate buffer (200 mM pH7.4) was addedand absorbance at 490 nm was recorded (Shimadzu UV-2102 PC, UV-visible Scanning Spectrophotometer). Two control treatments were used, without L-DOPA or without protein extract. Peroxidase activity was determined using 60 µl of protein extract, 210 µl of phosphate buffer (10 mM pH 6) and 240 µl of pyrogallol (Sigma P0381) with 150 µl hydrogen peroxide 1.6 volumes to activate the assay, after 3 min the absorbance was recorded at 470 nm. Control treatment containing 60 µl of protein extract and 600 µl of phosphate buffer (10 mM pH 6) was done. Phenoloxidase and peroxidase activities were expressed as the change in absorbance per mg protein (Abs mg protein-1). All assays were run by triplicate. Soluble protein content and enzymatic activity were analyzed with ANOVA after testing normality and homogeneity of variances. Significant differences were considered at p < 0.05. When significant differences were found, a Tukey-Kramer Multiple Comparison test was performed to locate these differences. Analysis were made using NCSS 8 Software. Results and Discussion The two sea anemone species used in this study, A. marplatensis and B. zamponii, demonstrated differing levels of constituent immunity, as indicated by the immune parameter activities. Soluble protein did not differ significantly among species and tissues, with levels between 15.8 and 20.2 mg ml-1 for A. marplatensis and 17.0 and 21.8 mg ml-1 for B. zamponii. Phenoloxidase (PO) activity was observed in all tissues evaluated, having the ectoderm of B. zamponii the highest PO activity at 0.025 Abs 470 nm mg protein-1. Mean PO activities of endoderm and tentacles for both species and ectoderm of A. marplatensis were significantly lower than B. zamponii's ectoderm (Figs 1a, c). Palmer et al. (2010) demonstrated that PO activity was present in different coral families, such as, Euphylidae, Acroporidae, Pocilloporidae, Alcyonacae, Merulinidae, Faviidae, Mussidae, Fungiidae, Poritidae and Oculinidae; and varied significantly among them. Phenoloxidase is the activating enzyme of the melanin-synthesis pathway, a key component of invertebrate immunity and the melanin-synthesis which provides cytotoxic defense, a protective barrier and structural support (Palmer et al., 2011). For anthozoans, melanization was the first documented within a sea fan, as a barrier against a fungal infection (Petes et al., 2003; Mullen et al., 2004) described the amebocytes involved. In the same species, aggregations of amebocytes were documented around fungal infections, and their granular content was confirmed to be melanin 193 Fig. 1 (a, b, c, d). Aulactinia marplatensis versus Bunodosoma zamponii tissue comparisons for mean (± s.e.) phenoloxidase and peroxidase activities, ect = ectoderm, end = endoderm, tent = tentacles. (Mydlarz et al., 2008). Tucker et al. (2011) demonstrated that amebocytes were commonly encountered in the mesoglea, in the thick fibril free zone under the epidermis of anemone Nematostella vectensis, while melanin-containing granular cells were located predominantly in the epidermis in 15 scleractinian species by Palmer et al. (2010), this observation could be explain the highest PO activity registered in ectoderm of B. zamponii in the current study. Invertebrates with low PO activity are more susceptible to disease and similarly, scleractinian corals are more susceptible to bleaching and disease, as recently documented in a wide range of coral families (Palmer et al., 2011). Differences in residual PO activity among coral families indicate physiological disparities that may have implications at an ecological scale in terms of disease resistance, with families having higher PO activity being more able to resist infection. This prediction is consistent with correlations found between PO activity and immunocompetence for numerous invertebrates (Palmer et al., 2010). According to previous information, we may be suggesting that B. zamponii is more able to resist infection, however additional research is necessary. The super-family of peroxidase enzymes contains many isoforms which partake in a variety of metabolic functions. In animal, peroxidase enzymes (PE) are involved in disease resistance and stress responses. Changes in peroxidase levels can signify immunomodulation due to contaminants and other environmental stressors (Mydlarz and Harvell, 2007). These authors examined the inducibility of coral peroxidases by experimentally exposing corals to fungal pathogen and found that enzyme activity was induced after an incubation period and they also hypothesize that Gorgonia ventalina utilizes the peroxidases as an integral component in disease resistance pathways. Palmer et al. (2011) 194 evidenced peroxidase activity in bleached and healthy colonies of Acropora millepora and proposed that this enzyme is potentially important for mitigate the effect of oxidative stress. Dikens and Shick (1984) established that peroxidase activity in the sea anemone Anthopleura elegantissima was highest in the tentacles and oral disc. A more detailed localisation of this enzyme was attemped by Hawkridge et al. (2000) who localised the antioxidant enzymes in granulated vesicles, accumulation bodies of endosymbiotic algae and all forms of cnida in the temperate sea anemone Anemonia viridis and tropical coral Goniopora stokesi, both species considered abundant in their respective habitats. In the current study, mean peroxidase activity was approximately equivalent (~0.06 Abs 470 nm mg protein-1) for all tissues of A. marplatensis and tentacles of B. zamponii, and significantly higher than ectoderm and endoderm of B. zamponii (~0.03 Abs 470 nm mg protein-1) (Figs 1b, d). Phenoloxidase and peroxidase activities are commonly associated with the mechanisms of innate immunity in invertebrates, and were confirmed for the first time in the studied actiniarians. The high production of phenoloxidase observed in B. zamponii would provide a continual level of resistance to infection and this species to be less susceptible to stress and disease, compared to A. marplatensis. However the latter has a possible additional defense, the attached cover of gravel on its column that could constitute a physical barrier to pathogens. This cover is common on the column of intertidal sea anemones with verrucae, and can support a small but rich community of invertebrates and algae (Barcellini, 2011). Thus, via collaborative efforts between ecologists, immunologists, cell biologists, and physiologists, we may expand the current understanding of innate immunity in naturally occurring ecologically important species. With greater understanding of the connections between environment and organismal immunity, we can make predictions about the effects of changing climate and environment on immunocompetence and disease outbreaks (Mydlarz et al., 2006). Acknowledgements This research was financially supported by the Mar del Plata National University (Argentina) Grant No. 585-12. We are also grateful to Lic. Yamila Rodriguez for her suggestions for the draft of this manuscript. References Acuña FH. Ecología trófica de actiniarios (Cnidaria, Anthozoa) intermareales: selección de la talla de las presas. Physis A53: 124-125, 1-5, 1997. Acuña FH, Zamponi MO. Ecology of intertidal sea anemones. Density, dispersion and autoecology of Phymactis clematis Dana, 1849 (Anthozoa, Actiniaria). Cienc. Mar. 21: 1-12, 1995a. Acuña FH, Zamponi MO. Feeding ecology of intertidal sea anemones (Cnidaria, Actiniaria): food sources and trophic parameters. Biociências 3: 73-84, 1995b. Acuña FH, Zamponi MO. Population structure and sex ratio of the intertidal sea anemone Phymactis clematis Dana, 1849 (Actiniaria: Actiniidae). Biociências 4: 3-16, 1996a. Acuña FH, Zamponi MO. Trophic ecology of the intertidal sea anemones Phymactis clematis Dana, 1849; Aulactinia marplatensis (Zamponi, 1977) and A. reynaudi (Milne-Edwards, 1857) (Actiniaria: Actiniidae): relationships between sea anemones and their preys. Cienc. Mar. 22: 397-413, 1996b. Acuña FH, Zamponi MO. The use of cnidocysts for ecological races identification from sea anemones populations (Anthozoa, Actiniidae). Iheringia, Sér. Zool. 82: 9-18, 1997. Acuña FH, Zamponi MO. Estructura poblacional de Aulactinia marplatensis (Zamponi, 1977) y Aulactinia reynaudi (Milne-Edwards, 1857) (Actiniaria: Actiniidae) en Argentina. Biociências 6: 13-33, 1998. Acuña FH, Excoffon AC, Zamponi MO. Hábitos alimenticios de las anémonas de mar (Actiniaria, Actiniidae) del Puerto de Mar del Plata (Argentina). Biociências 7: 155-158, 1999. Acuña FH, Excoffon AC, McKinstry SR, Martínez DE. Characterization of Aulactinia (Actiniaria: Actiniidae) species from Mar del Plata (Argentina) using morphological and molecular data. Hydrobiologia 592: 249-256, 2007. Arbeloa E, Carignan M, Acuña FH, Churio MS, Carreto JI. Mycosporine-like amino acid content in the sea anemones Aulactinia marplatensis, Oulactis muscosa and Anthothoe chilensis. Comp. Bioch. Physiol. 156B: 216-221, 2010. Barcellini M. Fauna y flora asociadas a la anémona de mar Aulactinia marplatensis (Zamponi, 1977) (Actiniaria, Actiniidae) de Mar del Plata y zonas adyacentes. Tesis de Grado de Licenciatura en Ciencias Biológicas, Facultad Ciencias Exactas y Naturales, Universidad Nacional de Mar del Plata, Mar del Plata, 2011. Braga Gomes P, Schama R, Solé-Cava AM. Molecular and morphological evidence that Phymactis papillosa from Argentina is, in fact, a new species of the genus Bunodosoma (Cnidaria: Actiniidae). J. Mar. Biol. Ass. UK 92: 895-910, 2012. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254, 1976. Dikens JA, Shick JM. Photobiology of the symbiotic sea anemone, Anthopleura elegantissima: defenses against photodynamic effects, and seasonal photoacclimatization. Biol. Bull. 167: 683-697, 1984. Excoffon AC, Zamponi MO. La biología reproductiva de Phymactis clematis Dana, 1849 (Actiniaria: Actiniidae): gametogénesis, períodos reproductivos, desarrollo embrionario y larval. Spheniscus 9: 25-39, 1991. Excoffon AC, Zamponi MO. Una excepción al patrón reproductivo del género Aulactinia Verrill, 1864 (Cnidaria: Actiniaria). Biociências 5: 207-217, 1997. 195 Hawkridge JM, Pipe RK, Brown BE. Localisation of antioxidant enzymes in the cnidarians Anemona viridis and Goniopora stokesi. Mar. Biol. 137: 1-9, 2000. Mullen K, Peters EC, Harvell, CD. Coral resistence to disease. In: Rosenberg E, Loya Y (eds), Coral health and disease, New York, pp 377- 399, 2004. Hutton DMC, Smith, VJ. Antibacterial properties of isolated amoebocytes from the sea anemone Actinia equine. Biol. Bull. 191: 441-451, 1996. Olano CT, Bigger CH. Phagocytic activities of the gorgonian coral Swifiiaexserta. J. Invertebr. Patol. 76: 176-184, 2000. Koljak R, Boutaund O, Shieh BH, Samel N, Brash AR. Identification of a naturally occurring peroxidase-lipoxygenase fusion protein. Science 277: 1994-1996, 1997. Palmer CV, Bythell JC, Willis BL. Levels of immunity parameters underpin bleaching and disease susceptibility of reef corals. FASEB J. 24: 1935- 1945, 2010. Matozzo V, Giacomazzo M, Finos L, Marin MG, Bargelloni L, Milan M. Can ecological history influence immunomarker responses and antioxidant enzyme activities in bivalves that have been experimentally exposed to contaminants? A new subject for discussion in "eco-immunology" studies. Fish Shellfish Immunol. 35: 126-35, 2013. Palmer CV, Bythell JC, Willis BL. A comparative study of phenoloxidase activity in diseased and bleached colonies of the coral Acroporamillepora. Dev. Comp. Immunol. 35: 1096-1099, 2011. Palmer CV, Mydlarz LD, Willis BL. Evidence of an inflammatory-like response in non-normally pigmented tissues of two scleractinian corals. Proc. R. Soc. Lond. 275: 2687-2693, 2008. Mydlarz, LD, Harvell, CD. Peroxidase activity and inducibility in the sea fan coral exposed to a fungal pathogen. Comp. Bioch. Physiol. 146: 54-62, 2007. Petes LE, Harvell CD, Peters EC. Webb MAH, Mullen KM. Pathogens compromise reproduction and induce melanization in Caribbean sea fans. Mar. Ecol. Prog. Ser. 264: 167-171, 2003. Mydlarz LD, Holthouse SF, Peters EC, Harvell, CD. Cellular responses in sea fan corals: granular amoebocytes react to pathogen and climate stressors. PloS One 3, e1811, 2008. Tucker RP, Shibata B, Blankenship TN. Ultraestructure of the mesoglea of the sea anemone Nematostella vectensis (Edwardsiidae). Invertebr. Biol. 130: 11-24, 2011. Mydlarz LD, Jones LE, Harvell, CD. Innate immunity, environmental drivers, and disease ecology of marine and freshwater invertebrates. Annu. Rev. Ecol. Evol. Syst. 37: 251-288, 2006. Zamponi MO, Excoffon AC. Algunos aspectos de la biología reproductiva de Bunodactis marplatensis Zamponi, 1977 (Actiniaria: Actiniidae). Spheniscus 4: 9-18, 1986. Mydlarz LD, Palmer CV. The presence of multiple phenoloxidases in Caribbean reef-building corals. Comp. Bioch. Physiol. 159: 372-378, 2011. 196