Polar Biology https://doi.org/10.1007/s00300-018-2346-x

ORIGINAL PAPER

Detection of peroxiredoxin‑like protein in Antarctic sea urchin (Sterechinus neumayeri) under heat stress and induced with pathogen‑associated molecular pattern from Vibrio anguillarum Byron Morales‑Lange1 · Marcelo González‑Aravena2 · Alejandro Font2 · Fanny Guzmán3 · Luis Mercado1,2,3  Received: 3 June 2017 / Revised: 30 May 2018 / Accepted: 1 June 2018 © Springer-Verlag GmbH Germany, part of Springer Nature 2018

Abstract Antarctic marine organisms have developed in an environment of low temperatures and high levels of stability. Consequently, these species have lost the ability to adapt to sudden changes in temperature. Rising ocean temperatures could make the Antarctic sea urchin Sterechinus neumayeri vulnerable to pathogens, triggering responses that increase oxidative stress. In order to understand how the immune system reacts, we can analyze the expression of anti-oxidant molecules such as the peroxiredoxins (Prxs). Prxs are an anti-oxidant protein family with conserved catalytic redox-active cysteine residues. In S. neumayeri, one full-length cDNA of the gene that encodes Prx (Sn-Prx) was characterized. The Sn-Prx cDNA contains a 786-bp open reading frame, which encodes 262 amino acids, including two conserved cysteine residues that are characteristic of the typical 2-Cys subgroup of the Prx family. An in silico analysis was performed to establish epitope molecules, which were then chemically synthesized and used to obtain antibodies. The antibodies were validated by indirect ELISA against a synthetic peptide and western blot against S. neumayeri proteins. In different tissues, the expression of Sn-Prx protein was increased after Vibrio anguillarum challenge. Heat stress also increased expression of Sn-Prx in coelomocytes after 7 days, but the availability of the protein decreased in digestive gland tissues after 2 and 3 weeks of heat stress. This may indicate the involvement of Prx in antioxidant response in S. neumayeri. The evidence presented in this study proposes anti-Prx antibody as an experimental evaluation tool that can be used to establish a baseline of the ability of S. neumayeri antioxidant response. Keywords  Heat stress · Sterechinus neumayeri · Prx · Anti-oxidant · Antarctica

Introduction The Antarctic environment, just like animal body fluid, has a high concentration of dissolved gases such as O ­ 2. This condition can increase the formation rate of reactive oxygen species (ROS) (Tolomeo et al. 2016). Additionally, heat * Marcelo González‑Aravena [email protected] 1



Grupo de Marcadores Inmunológicos en Organismos Acuáticos, Laboratorio de Genética e Inmunología Molecular, Instituto de Biología, Facultad de Ciencias, Pontificia Universidad Católica de Valparaíso, Avenida Universidad 330, Campus Curauma, Valparaíso, Chile

2



Laboratorio de Biorrecursos Antárticos, Departamento Científico, Instituto Antártico Chileno, Plaza Muñoz Gamero 1055, Punta Arenas, Chile

3

Nucleo Biotecnológico de Curauma, Av. Universidad 330, Campus Curauma, Valparaíso, Chile



stress is often associated with increased ROS generation, which leads to oxidative damage (Heise et al. 2003). Under normal conditions, ROS are produced by aerobic metabolism, and they play a specific role during immune responses and signal transduction. However, excessive accumulation of ROS can increase oxidative stress and may produce accumulative damage on a cellular level. Anti-oxidant enzymes, such as catalase, superoxide dismutase and peroxiredoxin, act together to limit the effects of oxidant molecules on cells and tissues (Tolomeo et al. 2016). Echinoderms have an integrated and complex innate immune system, with similarity to higher vertebrates. This innate system has cellular components, for example, coelomocytes which are able to trigger functions to fight infections, such as chemotaxis, phagocytosis and cytotoxicity, through reactive species, such as hydrogen peroxide and nitric oxide, which destabilize the pathogen and cause its destruction (Smith et al. 2010; Smith 2012). This increase in the production of ROS can be dangerous not only for

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pathogens but also for different host tissues. Another factor that can modify the immune response of echinoderms is heat stress. In the Antarctic sea urchin Sterechinus neumayeri, it has been described that the rise in the temperature of Antarctic waters (caused by global warming) makes it susceptible to attack by potentially pathogenic species (Branco et al. 2012; Silva 2013). This is because the increased temperature affects immune processes like phagocytosis and the spread capacity of immune cells (Branco et al. 2012). These two factors may influence cellular production of ROS, which can become a problem under any form of physiological hazard (Abele and Puntarulo 2004). Anti-oxidant proteins are part of the system that must regulate reactive species (Du et al. 2013). Peroxiredoxins (Prx) are a family of thiol-specific anti-oxidant proteins that are highly conserved in eukaryotes. These proteins have been identified in yeast, plant, and animal cells, and their primary location is in the cytosol, though they are also found in mitochondria, chloroplasts, and peroxisomes, in association with nuclei and membranes. All Prxs share the same basic catalytic mechanism, in which an active site cysteine (the peroxidatic cysteine) is oxidized to a sulfenic acid by the peroxide substrate. The recycling of the sulfenic acid back to a thiol is what distinguishes the three enzyme classes. Prxs are divided into three classes: typical 2-Cys Prxs; atypical 2-Cys Prxs; and 1-Cys Prxs (Wood et al. 2003). Depending on the subtype of Prx, the thiol reacting with the primary oxidation product (E-SOH) may be a cysteine residue of a second subunit (typical 2-Cys Prx), a cysteine residue of the same subunit (atypical 2-Cys Prx), or a reducing substrate (1-Cys Prx) (Trujillo et al. 2007). In addition to their peroxidase activity, members of the 2-Cys Prx subfamily appear to serve as peroxide sensors for other proteins and as molecular chaperones. During catalysis, the peroxidatic Cys–SOH of 2-Cys Prxs is occasionally further oxidized to Cys– SO2H before disulfide formation, resulting in inactivation of peroxidase activity. This hyperoxidation, which is reversed by the ATP-dependent enzyme sulfiredoxin, modulates the sensor and chaperone functions of 2-Cys Prxs (Rhee and Woo 2011). Prx gene families have been described in a variety of marine invertebrates such as the shrimps Fenneropenaeus chinensis (Zhang et al. 2007) and Marsupenaeus japonicus; these Prx belong to the typical class 2-Cys-Prx subfamily which includes four different isoforms (Prx1–Prx4) (Bacano-Maningas et al. 2008). Prx may act as cytoprotective enzymes against the high levels of ROS/RNS produced during inflammatory processes. They act as modulators of redox signaling involved in the control of inflammatory cell functions (Knoops et al. 2016). In other animals, Prx can have different roles, such as natural killer enhancer factor (Esteban et al. 2013), chaperones (Trujillo et al. 2007; Rhee and Woo 2011), or in thermal stress resistance (Oláhová et al. 2008; Park

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et al. 2008; Tolomeo et al. 2016). In sea urchins, the existence of Prx was predicted at the genetic level in Strongylocentrotus purpuratus, but the presence of this protein and its availability is unknown in S. neumayeri. To date, only atypical 2-Cys Prxs belonging to Prx6 have been identified in Antarctic animals, in the thermally stressed Antarctic bivalve Laternulla elliptica (Park et al. 2008) and the Antarctic fish Trematomus bernacchii (Tolomeo et al. 2016). In both these Antarctic marine species, levels of these genes were increased, suggesting that this atypical 2-Cys class of Prxs plays a protective role against oxidative stress caused by heat stress. The induction of Prx 1 and Prx 6 have also been observed during bacterial challenge, suggesting that these genes play a role in a protective response against bacterial infection. The main aim of this work is to characterize a Prxlike protein at the molecular level for the first time in S. neumayeri (Sn-Prx), and to demonstrate its availability at the protein level through the production of an antibody specific for this species. Thus the interest is in proposing a Prx-like protein as a biomarker in S. neumayeri under thermal stress, and induced with a pathogen-associated molecular pattern from Vibrio anguillarum.

Materials and methods Samples Individuals of S. neumayeri were collected in Fildes Bay (62°12′4.96″S, 58°57′45.50″W), King George Island (South Shetland Islands). Antarctic sea urchins with a diameter of 32 ± 2 mm were acclimated for 5 days at the Escudero Scientific Station (Chile). In order to conduct V. anguillarum challenges, individuals of S. neumayeri were divided into two pools (control and induced; 20 individuals per pool) with 10 L of UVsterilized and vacuum-filtered seawater (0.2-µm filter), with a constant regulated temperature of 1 °C. The induced group was injected with 1­ 07 heat-attenuated cells from V. anguillarum, while the control group was injected with sterile PBS. Samples were taken at 1, 3, 6, 21 and 26 h post-injection. To test for heat stress, individuals of S. neumayeri were heat-stressed at 3 °C, while the control group was maintained at 1 °C. The sampling times for the induced and control groups were 7, 14 and 21 days. For each sampling time, eight animals were sacrificed (four control and four induced), and individual digestive glands, esophagus, and coelomocytes (extract of coelomic fluid) were sampled.

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Characterization of peroxiredoxin sequence

Results

The cDNA sequence of a Prx-like peptide from S. neumayeri was obtained from the assembled contigs, generated from mRNA-seq in the digestive gland (Bioproject PRJNA376030). Sequence manipulation and alignments were produced and displayed in CLC Main workbench software (Qiagen).

Molecular characterization of Sterechinus neumayeri peroxiredoxin gene

Production and validation of antibodies Polyclonal antibodies were generated in CF-1 mice (4 weeks old) against a synthetic antigenic epitope for Sn-Prx. Antigenic epitopes were determined using a method described by Bethke et al. (2012) from the translated sequence of S. neumayeri (KU756581). Antigenic epitopes were then synthesized in accordance with Rojas et al. (2012). For antibody production, animals were subcutaneously injected at 1, 7 and 14 days with 300 μg immunogen diluted 1:1 in FIS as a T helper cell activator and 1:1 in Freund’s adjuvant (Thermo). The antiserum was collected on day 18, centrifuged at 700 g for 10 min and the supernatant was then stored at − 20 °C. Antibody affinity was determined by indirect ELISA (Morales-Lange et al. 2015) against the synthetic peptide of Prx, and antibody specificity was determined by SDS-PAGE followed by western blot analysis, as described previously in Schmitt et al. (2015), using a pool of four samples of induced organisms (coelomocytes and esophagus).

Detection of peroxiredoxin‑like protein in Sterechinus neumayeri Sn-Prx was analyzed in samples from digestive glands, esophagus and coelomocytes by indirect ELISA (MoralesLange et al. 2015). Briefly, each sample was diluted in Carbonate Buffer (NaHCO3 60 mM, pH 9.6) and seeded (by duplicate) in a 96-well plate at 35 ng µL−1 (100 µL) for overnight incubation at 4 °C. After blocking with 1% Bovine Serum Albumin for 2 h at 37 °C, the plates were incubated for 90 min at 37 °C with the first antibody anti-epitope of Sn-Prx (1:500). The second antibody-HRP (Thermo) was incubated for 1 h at 37 °C at 1:7000 dilution. Finally, chromagen substrate 3,3′,5,5′-tetramethylbenzidine single solution (Invitrogen) was added (100 µL) and incubated for 15 min at room temperature. The reaction was stopped with 50 µL of 1 N sulfuric acid and read at 450 nm on a VERSAmax microplate reader (Molecular Devices).

Data analysis Calculations of means and Student’s t test were carried out using GraphPad 6. Differences were considered significant at a p value < 0.05.

The full-length sequence of a putative Prx gene was represented by a single contig: C219866. This 2221-bp cDNA sequence of Sn-Prx contained a 786-bp coding region corresponding to 262 amino acids. The theoretical molecular weight of Sn-Prx, based on the deduced amino acid sequence, was calculated to be 28.926 kDa, with an isoelectric point (pI) of 4.88. Multiple sequence alignments of the deduced protein sequences of Sn-Prx with other invertebrate and vertebrate Prx sequences revealed two invariant cysteine residues, one near the N-terminus and one near the C-terminus, each followed immediately by a proline residue (Fig. 1a). The deduced protein sequence of Sn-Prx showed 82.5% protein sequence identities to the purple sea urchin Strongylocentrotus purpuratus and showed 60.9% protein sequence identities to the invertebrates Ixodes scapularis and Ciona intestinalis (Fig. 1b). In terms of structural domains, Sn-Prx shows a typical 2-Cys Prx subfamily domain (www.ncbi.nlm.nih.gov/cdd/) on the deduced amino acid sequence (interval 6–176). The Sn-Prx present region I and the CP loop, (x)DFTFVCPTE, and region II, D(T/S) (x)(F/Y)(x)H(xx)W(xx)(S/T/V), which are necessary for decamer formation.

Peptide design and synthesis In silico analysis of the deduced amino acid sequence indicates that the candidate peptide (VDETLRLVKAFQF) used to obtain antibodies is located from amino acids 150 to 163 of the obtained primary sequence. This region shows high antigenicity (http://imed.med.ucm.es/Tools​/antig​enic.pl), low hydrophobicity (Expasy, ProtScale) and has an alphāhelix secondary structure (Fig. 2). The results of the chemical synthesis show a simple peak in the HPLC profile at 6.5 min (Fig. 3a), and mass spectrometry shows the expected molecular weight (1565.8 Da) (Fig. 3b).

Production and validation of antibodies Through indirect ELISA, it was established that the antiSn-Prx peptide antibodies are able to recognize the peptide in proportion to its concentration (r = 0.9874; Fig. 4). To demonstrate recognition of the antibody for the complete molecule, a western blot was performed with a pool of samples of coelomocytes and esophagus induced with V. anguillarum and heat stress. These results (Fig. 5) show that the antibodies were able to recognize the protein at

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Fig. 1  Alignment and percentage identities of Sn-Prx sequence. a Multiple alignment of the amino acid sequence of the Sn-Prx sequence and Prx proteins from different invertebrates and vertebrates. The reactive cysteines and conserved Arg involved in the folded conformation of the Cp loop are indicated with an asterisk. The two regions involved in decamer formation are shown in the rectangles. Region I is part of the conserved loop–helix active-site motif and plays a key role in decamer formation by forming a sur-

face complementary to region II of the adjacent dimer. b Percentage of protein identities between several Prx. Accession numbers: Aplysia californica XP_005089483; Saccoglossus kowalevskii XP_002733995; Strongylocentrotus purpuratus XP_011663059; Homo sapiens Q06830; Notothenia coriiceps XP_010779546; Ixodes scapularis XP_002400518; Bos taurus NP_776856.1; Aedes aegypti AAL37254; Mus musculus NP_035693; Danio rerio NP_001002468

the expected weight for dimers (46 kDa) and monomers (23 kDa) in coelomocytes, and for the monomer (esophagus) of S. neumayeri challenged with V. anguillarum

(Fig. 5a). In the heat stress test, Sn-Prx recognition in coelomocytes and esophagus samples was for the expected

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6th hour of challenge, the detection again increases up to 1.20 (p < 0.05) over the control. At 21 h of challenge, SnPrx was similar to the control and, by the end of the kinetic experiment, it shows a significant increase to 1.47 (p < 0.05). The availability of Sn-Prx in the heat stress test (Fig. 6b) shows a significant rise in detection in coelomocytes (1.12) at 7 days of induction at 3 °C (p < 0.05). Availability of SnPrx then drops under that of the control at 14 days (0.86, p < 0.05) and 21 days (0.93). In esophagus tissue, Sn-Prx detection shows no significant differences compared to the control. In the digestive glands, Prx detection shows significant drops only at 21 days (0.59, p < 0.05).

Discussion

Fig. 2  Tridimensional representation (by Phyre2, Kelley et  al. 2015) of the primary structure of Sn-Prx, obtained from translated sequences (GenBank KU756581). Black indicates the antigenic peptide selected (VDETLRLVKAFQF) for synthetic synthesis and antibody production

weights of the tetramer (92 kDa) and pentamer (115 kDa) (Fig. 5b).

Availability of Sterechinus neumayeri‑peroxiredoxin‑like protein In the V. anguillarum challenge test, the availability of SnPrx by indirect ELISA (Fig. 6a) in coelomocytes changes with each passing hour post-induction in comparison to the control. One hour post-challenge, there is an increase of 1.29 in the detection (p < 0.05) of Sn-Prx, which rises to 1.35 by the third hour (p < 0.05). Six hours post-challenge, the detection falls, but is still above the control value (1.05), while at 21 h, the detection reaches the peak value of the series, with 1.41 (p < 0.05). Sn-Prx detection then begins falling, but remains higher than the control (1.04). In the esophagus, Sn-Prx does not show significant changes compared to control during hours 1, 3, 6 and 21. Only at 26 h of induction with pathogen-associated molecular patterns (PAMPs), is there a significant increase (1.08). In the digestive gland, a significant increase in the detection of Sn-Prx was observed over the control at 1 h of challenge (1.14). This detection drops by a third to 0.69 by 3 h of challenge. However, at the

Antarctic marine animals are exposed to high concentrations of dissolved gases like ­O2. This condition can increase the formation rate of ROS (Tolomeo et al. 2016) that results in oxidative damage to tissues and cells (Heise et al. 2003). Additionally, heat stress can increase ROS generation, and elevated water temperature can also act as a stressor, impacting the immune responses of marine invertebrates, potentially increasing their susceptibility to microbial infections as antibacterial activity becomes significantly depressed (Dang et al. 2012). Anti-oxidant enzymes such as Prx act to limit the effects of oxidant molecules on cells and tissues (Tolomeo et al. 2016). The Prx proteins are widely distributed in animals and possess different functions, as anti-oxidants, chaperones, enhancers of cytotoxic cells and thermal resistance proteins (Wood et al. 2003; Trujillo et al. 2007; Oláhová et al. 2008; Rhee and Woo 2011). Although Antarctic marine species are more sensitive to temperature variations than other regional marine groups, the consequences of climate change in marine invertebrates remains poorly understood (Peck et al. 2004; Branco et al. 2012). S. neumayeri is a good candidate for a study model for Antarctic subjects because it is the most abundant sea urchin in shallow Antarctic waters; it shows circum-Antarctic distribution and plays an important role in the benthic trophic web (Brey et al. 1995). Additionally, sea urchins have historically been used as classic experimental models due to their evolutionary proximity to higher organisms (Smith et al. 2010; Ghosh et al. 2011; Majeske et al. 2013). Their use has allowed advances in gene regulation, molecular embryology, fertilization, cell biology, evolution, population genetics, toxicology, and immunology (Smith et al. 2010; Ghosh et al. 2011). However, little information is available regarding the molecules involved in the immune response of S. neumayeri. The characterization of the Prx sequence in S. neumayeri at a molecular level shows a typical 2-Cys Prx subfamily conserved domain. This is confirmed by the presence of two

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Fig. 3  Peptide synthesis (P1708). a High-performance liquid chromatography. b Mass spectrometry

Fig. 4  Peptide recognition by the antibody produced by indirect ELISA. OD optical density at 450 nm, R Pearson correlation coefficient

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cysteine residues, one near the N-terminus and one near the C-terminus in this molecule, which are conserved in comparison with a wide range of animals. These results allow us to propose that the catalytic function of the Prx enzyme could be conserved in this organism. In terms of structural regions, two regions involved in decamer formation are conserved. This may be associated with functions as chaperones under oligomeric conformations (Rhee and Woo 2011). The presence of Prx of S. neumayeri with 82.58% identity with the Prx sequence of Strongylocentrotus purpuratus, and with homology with other invertebrates such as Ixodes scapularis and Ciona intestinalis, in addition to the report of Prx in Antarctic marine organisms (Park et al. 2008; Tolomeo et al. 2016), follows the idea that Prx is present transversally in different animals (Wood et al. 2003). The lack of antibodies for the characterization of molecules in aquatic organisms has been a historical problem for

Polar Biology Fig. 5  Prx detection by western blot. a S. neumayeri challenged with pathogen-associated molecular patterns from V. anguillarum. b S. neumayeri under heat stress. C coelomocytes, E esophagus. c Negative control without anti-Prx antibody

Fig. 6  Prx detection in fold change relative to control by indirect ELISA. a S. neumayeri challenged with PAMPs from V. anguillarum (n = 4 for each bar). b S. neumayeri under heat stress (n = 4 for each bar). Asterisk represents significant differences (p < 0.05)

the evaluation of markers on a phenotypic level (Tafalla et al. 2013). In Antarctic organisms, the proposed existence of Prx has followed genetic and transcriptomic strategies (Park et al. 2008; Tolomeo et al. 2016). In the present study, we are proposing a characterization approach that incorporates phenotypic evidence, thus reliably establishing the presence of a molecule within a biological system.

The production and validation of polyclonal antibodies against synthetic peptides of Sn-Prx have proven a useful tool for the generation of immune markers able to recognize phenotypic parameters in S. neumayeri in the potential responses to challenges such as PAMPs and heat stress. This strategy has already been reported in the literature, where Bethke et al. (2012), Rojas et al. (2012) and Schmitt et al. (2015) use in silico sequence analysis, synthesis of epitopes, and generation of antibodies against proteins of aquatic organisms, obtaining an effective tool for the characterization of the organism’s response to challenge at a phenotypic level. Sn-Prx detection in biological samples of S. neumayeri by antibodies against synthetic epitopes is the first report of this molecule at the protein level in sea urchins, which has only been predicted at the genetic level in the purple sea urchin (S. purpuratus). Temporal variation in the detection of Sn-Prx challenged with V. anguillarum shows a detection peak at 21 h postinfection in coelomocytes. It is described that Prx may act as cytoprotective enzymes against high levels of ROS/RNS produced during inflammatory processes. These proteins act as modulators of the redox signaling involved in the control of inflammatory cell functions (Knoops et al. 2016). In sea urchins, coelomocytes are inflammatory cells and are mainly involved in the execution of the immune response by recognizing PAMPs, phagocytosis, destruction by reactive species production and anti-oxidant regulation (Smith et al. 2010; Smith 2012). Therefore, increasing the availability of Sn-Prx in coelomocytes provides evidence to propose that S. neumayeri regulates the highly damaging molecules, increasing the availability of anti-oxidant proteins. Due to the toxicity of the reactive species, they should be degraded quickly and efficiently in order to prevent damage at the cellular level such as tissue (Heck et al. 2000). The detection of Prx in tissues such as esophagus and the digestive gland agrees with other studies that tissues can produce anti-oxidants in other sea urchins (Du et al. 2013), but it is the coelomocytes, that when activated, which migrate to the tissues such as esophagus and digestive gland and infiltrate the submucous regions, because the coelomocytes are mobile cells capable

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of infiltrating tissues in favor of developing a robust and integrated immune response to molecular patterns associated with pathogens (Gross et al. 1999; Borges et al. 2002; Branco et al. 2012). This process could explain the later sampling results (26 h), where Sn-Prx detection was increased in the tissues but falls in the coelomocytes, because the cells that remain in the coelomic fluid were not activated, and no increased availability of Sn-Prx was detected. Under heat stress, the detection of Sn-Prx shows an increase in the availability of this protein in coelomocytes induced for 7 days at 3 °C, but when the thermal stress is maintained for 14 and 21 days, there is a significant decrease in the availability of Sn-Prx relative to the control (S. neumayeri maintained at 1 °C) in coelomocytes (at 14 days) and in tissues like the digestive gland (at 21 days). This may be because S. neumayeri is a stenothermal organism that lives in a constant environment (Peck 2005), and heat stress reduces its capacity to control oxidative damage using antioxidative proteins as is done by other Antarctic invertebrates such as Yoldia eightsi (Abele et al. 2001). This species, when challenged by heat stress for a long time, decreases the availability of anti-oxidant enzymes such as superoxide dismutase. The rise of ocean temperatures due to global warming modifies the physical parameters of the water, and these changes affect populations of marine Antarctic invertebrates (Silva 2013), making them susceptible to potential pathogens (Branco et al. 2012) and oxidative damage (Abele and Puntarulo 2004). In other Antarctic animals under heat stress, Prx has been evaluated at the transcriptional level. In Laternula elliptica (Antarctic bivalve), which were induced at 10 °C for up to 48 h, Prx showed an increase of Le-PrxV and Le-PrxVI. This was associated with the role of Prx in controlling oxidative and heat stress (Park et al. 2008). Tolomeo et al. (2016), working with Trematomus bernacchii, showed that Prdx6B increases significantly against heat stress up to 5 °C, postulating that this fish would be tolerant to increased temperatures due to the increase in Prx. The decrease in Prx in S. neumayeri can mean low tolerance to the increasing temperatures of Antarctic waters, a reason why it would be a good study model in research related to global climate change. Sn-Prx detection by western blot showed different recognition levels between samples from the V. anguillarum challenge and the heat stress test, which may represent different functions of Sn-Prx. It has been reported in vertebrates that Sn-Prx has alternative functions depending on its molecular form. The monomer is an inactive form, the dimer has a catalytic function, and the oligomer form may act like chaperones (Wood et al. 2003; Kang et al. 2005). Our results show that, in the V. anguillarum challenge, coelomocytes have the dimer form and possess an anti-oxidant capacity, probably because the immune system is activated and the individuals

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need to control the reactive species. The dimer formation could be formed because the Sn-Prx has two redox-active cysteines, one attacking the peroxide substrate (ROOH) and becoming oxidized, and the second one attacking the cysteine-sulfenic acid to release water and form a disulfide bond (Wood et al. 2002). On the other hand, during the thermal stress, the oligomer form was detected, which can be related to the presence of two regions involved in decamer formation, since persistent stress causes the oligomerization of Prx for chaperone function. The Sn-Prx enzyme could be primarily redox-sensitive, with the reduced enzyme strongly favoring the (α2)5 decamer, and with the oxidized enzyme forming a mixture of predominantly lower order oligomeric assemblies (Wood et al. 2002). In the case of S. neumayeri, the tetramer and pentamer form could be favored by the redox state because the thermal stress produce an increase of respiration rate. In summary, the anti-polyclonal antibodies produced to recognize synthetic epitope molecules are able to recognize the peroxiredoxin-like protein at the expected weight in S. neumayeri samples. It was determined that, in response to V. anguillarum, coelomocytes, esophagus and digestive gland tissues increase the availability of Prx. At the same time, under heat stress, there is a decrease in the availability of this protein at 14 and 21 days. The molecule detected by the anti-epitope antibody could be used as a biomarker of antioxidant response in S. neumayeri under different challenges, consolidating this species as a working model to study Antarctic issues. Acknowledgements  This study was supported by Fondo Nacional de Desarrollo Científico y Tecnológico 1131001 (2013–2015). BM is a fellow of Advanced Human Capital Formation of CONICYT, Chile (21151176).

Compliance with ethical standards  Conflict of interest  The authors declare that they have no conflict of interest. Ethical approval  All procedures performed in studies involving animals were in accordance with the ethical standards of the institution or practice at which the studies were conducted.

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