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HSP70 from the Antarctic Sea Urchin Sterechinus Neumayeri: Molecular

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HSP70 from the Antarctic Sea Urchin Sterechinus Neumayeri: Molecular HSP70 from the Antarctic sea urchin Sterechinus neumayeri: molecular characterization and expression in response to heat stress Marcelo Gonzalez-Aravena, Camila Calfio, Luis Mercado, Byron Morales-Lange, Jorn Bethke, Julien de Lorgeril, Cesar Cárdenas To cite this version: Marcelo Gonzalez-Aravena, Camila Calfio, Luis Mercado, Byron Morales-Lange, Jorn Bethke, etal.. HSP70 from the Antarctic sea urchin Sterechinus neumayeri: molecular characterization and expres- sion in response to heat stress. Biological Research, Sociedad de Biología de Chile, 2018, 51, pp.8. 10.1186/s40659-018-0156-9. hal-01759214 HAL Id: hal-01759214 https://hal.archives-ouvertes.fr/hal-01759214 Submitted on 5 Apr 2018 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. González-Aravena et al. Biol Res (2018) 51:8 https://doi.org/10.1186/s40659-018-0156-9 Biological Research RESEARCH ARTICLE Open Access HSP70 from the Antarctic sea urchin Sterechinus neumayeri: molecular characterization and expression in response to heat stress Marcelo González‑Aravena1* , Camila Calfo1, Luis Mercado2, Byron Morales‑Lange2, Jorn Bethke2, Julien De Lorgeril3 and César A. Cárdenas1 Abstract Background: Heat stress proteins are implicated in stabilizing and refolding denatured proteins in vertebrates and invertebrates. Members of the Hsp70 gene family comprise the cognate heat shock protein (Hsc70) and inducible heat shock protein (Hsp70). However, the cDNA sequence and the expression of Hsp70 in the Antarctic sea urchin are unknown. Methods: We amplifed and cloned a transcript sequence of 1991 bp from the Antarctic sea urchin Sterechinus neu- mayeri, experimentally exposed to heat stress (5 and 10 °C for 1, 24 and 48 h). RACE-PCR and qPCR were employed to determine Hsp70 gene expression, while western blot and ELISA methods were used to determine protein expression. Results: The sequence obtained from S. neumayeri showed high identity with Hsp70 members. Several Hsp70 family features were identifed in the deduced amino acid sequence and they indicate that the isolated Hsp70 is related to the cognate heat shock protein type. The corresponding 70 kDa protein, called Sn-Hsp70, was immune detected in the coelomocytes and the digestive tract of S. neumayeri using a monospecifc polyclonal antibody. We showed that S. neumayeri do not respond to acute heat stress by up-regulation of Sn-Hsp70 at transcript and protein level. Further‑ more, the Sn-Hsp70 protein expression was not induced in the digestive tract. Conclusions: Our results provide the frst molecular evidence that Sn-Hsp70 is expressed constitutively and is non- induced by heat stress in S. neumayeri. Keywords: Hsp70, Coelomocytes, Antarctica, Echinoderms, Climate change Background [1]. In the last few decades, the Antarctic Peninsula has Te success of any organism relies on its niche adaptation shown a strong increase in temperature [2, 3]. Particu- and also on its ability to survive to environmental vari- larly, the increase of shallow seawater temperature could ation (e.g. maintaining homeostasis in stress situations). result in a negative impact on various stenothermal spe- In this regard, the induction of heat shock proteins (e.g. cies [4]. Sudden changes in sea surface temperature and Hsp70) has been widely reported across diferent taxa salinity has been reported in Potter Cove (King George Island) [5] and also in some areas of the Antarctic Pen- insula [6]. Recently, high mortality of krill has been *Correspondence: [email protected] reported near to Carlini Station related to signifcant 1 Laboratorio de Biorrecursos Antárticos, Departamento Científco, increases of particulate matter of glacial origin produce Instituto Antártico Chileno, Plaza Muñoz Gamero 1055, Punta Arenas, Chile by increased temperature [7]. Under this scenario, it has Full list of author information is available at the end of the article been experimentally determined that Antarctic marine © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. González-Aravena et al. Biol Res (2018) 51:8 Page 2 of 10 invertebrates may not survive to temperatures between modifcation in the adhesion and spreading capacity of + 5 and + 10 °C. Previous work suggests that ffty percent coelomocytes [27]. A recent study showed that several of the population of clams (Laternula elliptica) and lim- heat shock proteins could be expressed in coelomocytes pets (Nacella concinna) cannot carry out essential func- and digestive gland of S. neumayeri [28]. Tis work also tions between 2 and 3 °C, while individuals of the scallop showed that the expression of Hsp70 was not induced in Adamussium colbecki are unable to swim at 2 °C [4, 8]. response to water temperatures at 3 and 5 °C. However, Te thermal stress in S. neumayeri is characterized when no information about gene characteristics and protein the sea urchins ceased moving, relax the large spines. expression of Hsp70 from S. neumayeri was available. Tis was considered to be the critical thermal maximum Te aim of this study was to characterize the heat shock in sea urchins [9]. protein Hsp70 gene and construct and test a new anti- Te exposure of an organism to heat stress normally body for protein expression analysis in the coelomocytes induces a transient expression of heat stress proteins of S. neumayeri, and to determine whether its expression when the protein damage is detected. However, some is modulated after heat stress. Antarctic marine species, which have developed in an environment characterized by low temperatures and high Methods stability, have lost the ability to adapt to sudden changes Acute heat stress and sample collection in water temperature [10]. Tis situation has been dem- Sterechinus neumayeri individuals were hand collected onstrated in two species of Antarctic marine inverte- by SCUBA divers from 10 to 20 m depth in Maxwell Bay, brates (Odontaster validus and Paraceradocus gibber) Fildes Peninsula, King George Island (62º12′S–58º57′W), which were incapable of up-regulating the expression of during February 2012. Sea urchins were acclimated in a Hsp70 at 2 and 6 °C [11]. Nevertheless, it has also been cold chamber for 1 week at 0.5 °C. Tey were then trans- determined that some species of Antarctic mollusks such ferred directly to the respective aquarium at the selected as L. elliptica and N. concinna, are able to generate Hsp70 temperature (5 and 10 °C) and maintained for 48 h. Dur- during heat stress at 10 and 15 °C [12]. In L. elliptica ing the heat stress four sea urchins for each condition and N. concinna the GRP78 (glucose regulated protein, were recovered at 1, 24 and 48 h and the coelomic fuid 78 kDa) is expressed constitutively and in some tissues, was pooled immediately. Tree independent experiments could be induced by increased temperature [13]. were performed with the same experimental conditions. Te sea urchin Sterechinus neumayeri is an impor- After heat stress exposure the coelomic fuid was tant member of benthic communities, playing a key role collected into sterile tubes by cutting the peristomial in Antarctic marine ecosystems [14, 15]. In general, sea membrane. To avoid aggregation of coelomocytes, the urchins are considered a good model species for study- coelomic fuid was collected in the sterile tubes contain- ing the efects of environmental stressors. Sea urchin ing the modifed Alsever’s solution [29]. Coelomocytes embryos and coelomocytes have been used as indicators and tissues samples (digestive tract, esophagus, and axial in ecotoxicology studies [16]. In particular, increases in organ) were kept in RNALater (AMBION). After over- the number of coelomocytes, especially the red cells type, night incubation at 4 °C, they were placed at − 20 °C until have been documented in many studies reporting difer- used. ent kinds of stress like as injury on skeleton and dermis or environmental contamination by metals, oil solu- RNA extraction and cDNA synthesis ble fraction and industrial residues [17–20]. Terefore, Total RNA was extracted from several tissues (coelo- these cells could be used as stress markers to evaluate the mocytes, digestive tract, esophagus and axial organ) expression of a classical stress protein, such as Hsp70 [16, using a directly Trizol reagent (Invitrogen, USA) and 17]. Several studies have been conducted on S. neumayeri treated after with DNAse Turbo (Ambion) according to to evaluate environmental stressors including increased the manufacturer’s instructions. Concentration of total temperature, UV-B radiation, salinity, low pH [21–23]
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