The effect of thermal stress on protein composition in dogwhelks (Nucella lapillus) under normoxic and hyperoxic conditions

https://doi.org/10.1016/j.cbpa.2007.08.034Get rights and content

Abstract

In this laboratory study, dogwhelks (Nucella lapillus) were collected from the intertidal zone and exposed to 16 °C (ambient), 26.5 °C and 30 °C under normal and hyperoxic conditions respectively. It was shown that there was no thermally induced mortality at 26.5 °C, but that the mortality rate was 40–50% in 30 °C. This mortality rate was reduced to 10% if extra oxygen was provided, indicating that oxygen supply was setting the limit for whole organism thermal tolerance. Tissue samples were then analysed for protein features using two-dimensional gel electrophoresis, and both up and down regulation of proteins were visualised by silver staining and crosswise comparisons of gels from control vs. treated animals. The results clearly show that the protein profiles from dogwhelks exposed to increased water temperatures differ from those of the control, but that increased oxygen availability alleviates these differences thus increasing the similarity between heat-shocked and control animal protein pattern. This implies a more stable protein metabolism and might explain the increased survival of heat-shocked individuals when extra oxygen is supplied.

Introduction

Tolerance of fluctuating temperatures is of major concern to all organisms and of fundamental importance to intertidal organisms in particular. Responses to thermal changes occur at all levels of biological organization ranging from protein expression to organismal behaviour (Angilletta et al., 2006). The relationship between these levels is complex and molecular responses are not necessarily predictable of individual effects (Angilletta et al., 2006). It has been hypothesized that, in complex metazoans, the underlying problem with high temperatures is not only the temperature per se, but rather the reduced oxygen supply that occurs. In Pörtner et al. (2000) and Pörtner, 2001, Pörtner, 2002 it is suggested that the temperature tolerance is not set by responses at the cellular level, but rather limited by the ventilatory and circulatory system. As lethal temperature limits are approached, a transfer to anaerobic metabolism has been identified, and this transfer has been termed the “critical temperature” (Zielinski and Pörtner, 1996, Pörtner et al., 1998, Pörtner et al., 2000, Pörtner et al., 2006, Peck et al., 2002).

The intertidal gastropod Nucella lapillus is one of the most commonly used biomonitors in Europe and North America. In a study by Davenport and Davenport (2005), a comparison of median upper lethal temperature (MULT) amongst ten invertebrate, intertidal, species was performed. The dogwelks displayed large differences in MULT between individuals living on the lower shore and those living at the upper limit of shore distribution, with animals collected in the upper littoral zone having higher MULT values.

More recently, Davenport and Davenport (2007) investigated the relationship between oxygen supply and MULT in middle shore dogwhelks. They showed that increased oxygen supply resulted in a significant increase in MULT (and vice versa), a result consistent for this species with the synthesis of Pörtner (2001). It should be noted that N. lapillus commonly occurs in intertidal rock pools, and is consequently exposed to both hypoxia and hyperoxia in its natural environment (Morris and Taylor, 1983).

In this study we have extended the work on N. lapillus by applying a proteomic approach to the study of the relationship between oxygen supply and upper lethal temperature. Studies of large numbers of proteins in organisms, the proteome, mainly have a history of use in pharmaceutical investigations, but are now increasingly being used to understand key molecules involved in normal physiological pathways, as well as being diagnostic tools in environmental research (Shrader et al., 2003, Olsson et al., 2004). The principle behind this approach is that every organism:environment combination results in a unique set of organismal proteins, hence the set of proteins will depend on the type and amount of stress the organism has experienced. In order to elucidate the importance of such a protein response it is vital to couple molecular analyses with whole animal physiology.

In the present study we analyzed and compared the specific pattern of total protein expression in the survivors of different temperature:oxygen treatments. Our main aim was to study whether the observed phenomenon of increased survival rates at higher temperatures when oxygen concentrations are higher (Davenport and Davenport, 2007) corresponds with a more stable protein expression. If so, their protein pattern would resemble that of control animals (normal temperature, normoxia) more closely than would the protein pattern of animals exposed to high temperature without oxygen addition (normoxia). This would be the expected result if hyperoxia i.e. increased intracellular oxygen availability reduced or counteracted the effects of thermal stress.

Section snippets

Collection of specimens

N. lapillus (240 specimens) were collected from a single shore at Bullens Bay, Co. Cork, Ireland (N51°38.584′ W8°33.052′) in June 2004. Animals were collected from the middle of their vertical distribution: Davenport and Davenport (2005) showed significant differences in thermal tolerances of N. lapillus taken from the upper and lower distributional limits (perhaps reflecting the highly localized genetic substructuring already demonstrated for the species; Kirby, 2000). They were placed in an

Survival of the animals

During the exposures no mortalities occurred in the controls (16 °C) or in the 26.5 °C treatment. In 30 °C mortalities were observed and mortality rates were estimated to be 40–50% under normoxic conditions, but only 10% if oxygen availability was enhanced (hyperoxic conditions). These results are in agreement with those shown by Davenport and Davenport (2007).

Protein expression signatures in different temperature and oxygen regimes

The numbers of protein spots derived from the PDQuest analyses are demonstrated in Venn diagrams in Fig. 3. The protein expression

Discussion

This study, in accordance with Davenport and Davenport (2007), clearly shows that mortality induced by temperature stress is reduced under hyperoxia. A reversed scenario was observed in a study by Metzger et al. (2007), where elevated CO2 concentrations reduced arterial oxygen tension and thermal tolerance which resulted in induced mortality. The present study, as well as the findings by Metzger et al. (2007), is in line with the concept of oxygen and capacity-limited thermal tolerance (Pörtner

Acknowledgements

This study was funded by Stockholm Marine Science Centre (SMF) and Helge Ax:son Johnsons stiftelse. We also wish to thank Megan Fitzpatrick, Department of Biology, University of Maryland, for guidance in the proteomic laboratory.

References (29)

  • AngillettaM.J. et al.

    Coadaptation: a unifying principle in evolutionary thermal biology

    Physiol. Biochem. Zool.

    (2006)
  • BradleyB.P.

    Stress proteins: their detection and uses in biomonitoring

  • BradleyB.P. et al.

    Complex mixture analysis using protein expression as qualitative and quantitative tool

    Environ. Toxicol. Chem.

    (1994)
  • CherkasovA.A. et al.

    Temperature-dependent effects of cadmium and purine nucleotides on mitochondrial aconitase from a marine ectotherm, Crassostrea virginica: a role of temperature in oxidative stress and allosteric enzyme regulation

    J. Exp. Biol.

    (2007)
  • Cited by (23)

    • Seasonal proteome variation in intertidal shrimps under a natural setting: Connecting molecular networks with environmental fluctuations

      2020, Science of the Total Environment
      Citation Excerpt :

      Proteomic studies enable the detection of molecular markers that play a role in processes such as acclimation, adaptation and detoxification of contaminants, providing insights into the success of a species (Dalziel and Schulte, 2012; Hollywood et al., 2006; Joyce and Palsson, 2006). To date, field studies using physiological or biomarker approaches (e.g. Hofmann and Somero, 1995; Madeira et al., 2017; Padmini and Geetha, 2009; Shaw et al., 2004) and experiments using transcriptomics and proteomics (e.g. Fields et al., 2012; Gardeström et al., 2007; López et al., 2002; Stillman and Tagmount, 2009; Tomanek, 2011, 2014; Tomanek and Zuzow, 2010) have shown that the thermal limits and metabolic rates of intertidal, oceanic and freshwater species vary according to fluctuations in abiotic factors (e.g. temperature, salinity, oxygen). Transcripts and proteins related to protein folding, proteolysis, cytoskeletal dynamics, ribin, extension-like genes, immune response, cell growth, gene expression, rRNA synthesis, protein synthesis, energy metabolism, oxidative stress metabolism, extracellular matrix dynamics and structural and muscle proteins have been shown to change with environmental variation.

    • Oxygen-limited performance of the intertidal sea urchin Colobocentrotus atratus when submerged

      2018, Journal of Experimental Marine Biology and Ecology
      Citation Excerpt :

      One method of testing the OCLTT hypothesis is to determine whether experimentally increasing external levels of O2 can “rescue” performance of organisms under thermally stressful conditions (Pörtner et al., 2006). Rescue experiments have been performed in a range of taxa including Antarctic bivalves (Peck et al., 2007; Pörtner et al., 2006), temperate gastropods (Bjelde et al., 2015; Gardeström et al., 2007; Vosloo et al., 2013), crustaceans (Bowler, 1963), and fish (Bagherzadeh Lakani et al., 2013; Berschick et al., 1987; Foss et al., 2003; Mark et al., 2002). In one example of rescue experiments with intertidal invertebrates, Bjelde et al. (2015) showed that oxygen enrichment increased maximum heart rate (but not cardiac breakpoint or flat-line temperature) in the temperate intertidal limpet Lottia digitalis.

    • Ocean warming alters cellular metabolism and induces mortality in fish early life stages: A proteomic approach

      2016, Environmental Research
      Citation Excerpt :

      Therefore, proteomic research applied to ecology is still in its infancy and may unravel new pathways that play a role in adaptation processes (Karr, 2008; Dalziel and Schulte, 2012). The few proteomic studies performed on marine organisms have shown that several pathways are affected by temperature including cytoskeletal dynamics, energetic metabolism, oxidative stress metabolism, chaperoning activity, immune response, transcriptional regulation, protein turnover and signal transduction (e.g. López et al., 2002; Gardeström et al., 2007; Tomanek and Zuzow, 2010; Tomanek, 2011; Fields et al., 2012; Garland et al., 2015). The aims of this study were to assess the sensitivity of larval fish to ocean warming and extreme events in a highly commercial species, the sea bream Sparus aurata (Linnaeus, 1758) (IUCN Red List – Least Concern).

    • The use of -omic tools in the study of disease processes in marine bivalve mollusks

      2015, Journal of Invertebrate Pathology
      Citation Excerpt :

      A modest number of studies in bivalves are dedicated to the characterization of specific functions such as shell formation, reproduction, and development (e.g. Diz et al., 2012, 2013; Huan et al., 2012; Marie et al., 2012, 2011; Zhang et al., 2012). Most proteomic approaches in bivalves are mainly focused on studying responses to environmental stressors such as heavy metals (Huan et al., 2011; Muralidharan et al., 2012; Thompson et al., 2012), temperature (Fields et al., 2012; Gardeström et al., 2007; Pörtner, 2010), organic pollutants (Mi et al., 2007; Malécot et al., 2013; Olsson et al., 2004) and oxidative stress (McDonagh et al., 2005). Proteomic studies dedicated to the understanding of the response of marine bivalve species to disease are limited to the use of 2-D PAGE coupled with MS to identify differentially expressed spots and have relatively low throughput.

    View all citing articles on Scopus
    View full text