Adaptation and stress defence in interidal and subtidal Antarctic limpets (Nacella concinna): A Study of the plasticity of molecular and biochemical stress response in Antarctic invertebrates

The Antarctic limpet Nacella concinna is the most conspicuous macro-benthic species in the intertidal and splash zone of the maritime Antarctic. Larval recruitment in this non-homing limpet takes place in the subtidal, and part of the population then migrates to the intertidal in the beginning of spring. The migratory limpets belong to an ecomorph with a higher and heavier shell (left picture, limpets at the intertidal zone), whereas shells of permanently subtidal limpets are lighter and flatter (right picture, taken at the subtidal). The two ecomorphs can be found in every location along the peninsulas of the South Shetlands.

We are interested in the functional biodiversity of the two Nacella ecomorphs at Jubany station/Dallmann laboratory on King George Island, and investigate the behavioural and ecophysiological differences in-situ, and following exposure to air/emersion and to hypoxia. A special interest is on the expression and function of hypoxia inducible factor 1 (HIF-1) in the limpet model. This transcription factor is a universal master regulator of hypoxic metabolism and centrally involved in the metabolic regulation of cells and tissues under oxygen shortage. In the frames of the project we have obtained a first molluscan HIF-1-alpha sequence which, if compared to C. elegans and crustacean genes indicates rather high sequence variability to exist between invertebrates, in contrast to low variability among vertebrate genes.

Our results thus far demonstrate significant differences between limpet ecomorphs, especially in their response to stress. The intertidal limpets can control water loss during air exposure. They rely on air breathing, a behaviour known from intertidal limpets of temperate coasts and, in contrast to subtidal specimens, do not accumulate lactate, succinate or propionate. Instead they switch to acetate fermentation as additional energy source during prolonged air exposure. Once again submersed by the tides, they actively ventilate the water in their shells, reaching PO₂ values as high as 14 kPa, whereas the PO₂ values in the shell during air exposure rarely reach over 1 kPa.

Also, gills of intertidal limpets are significantly better protected against oxidative damage by the antioxidant enzymes catalase (CAT) and superoxide dismutase (SOD). In the digestive gland the subtidal animals had similar CAT and higher SOD activities, which might be protective against possible oxygen radical formation from high iron concentrations in subtidal animal digestive glands. After our second expedition that ended in the beginning of January 2008, we are currently finishing the biochemical analyses in air and hypoxia exposed limpets, and also obtained first results of HIF-expression under hypoxic stress in both limpet groups.

We further work on the Western blots with a new antibody produced to detect limpet HIF-1-alpha and on the publication of the data.

Summary (of the results)

The project compared the physiological adaptation of intertidal and subtidal subpopulations of the Antarctic limpet Nacella concinna with respect to stress response and the capacity to perform metabolic reorganization. We studied the metabolic stress response during desiccation and hypoxia exposure in intertidal N. concinna and in their subtidal congeners. It results that the limpets of the migratory subpopulation that colonize the intertidal mostly during the summer months, have developed many physiological and behavioural adaptations to cyclic air exposure. These involve delay of the onset of anaerobic metabolism and, apparently, on-switch of hypoxic gene regulation after prolonged air exposure of 12h. As marker for the activation of hypoxic genes, we analyzed the protein expression of hypoxia inducible factor HIFα in limpets. The limpet gene (see below) displays clear characteristics of HIFα, but has not evolved all important domains of mammalian HIFα genes. HIF protein levels depend on tissue oxygen levels and modulation of HIF stability by anaerobic metabolites (fumarate and succinate) is possible. HIF is an ancient transcription factor that can be used as a marker for stress response also in marine invertebrates.



Fig.1: HIF-α protein sequence with domains deduced from cDNA sequence of Nacella concinna. bHLH: basic helix-loop-helix domain, PAS A and PAS B: Per-ARNT-SIM, ODD: oxygen dependent degradation domain, CTAD: C-terminal transactivation domain. Black bars indicate functional hydroxylation sites. Motifs in front of the proline residues are written below the sequence. Striped box indicates the theoretical position of the N-TAD. Numbers below the sequence indicate the position of amino acid.

The Antarctic limpet is an interesting model organism to study phenotypic adaptations for survival in ecologically demanding environments, in this case the West Antarctic Peninsula intertidal. Climatic change is already becoming manifest in these regions, and if we want to document these changes on species, community and system level, we have to do it now.

Scientists

Doris Abele
Alfred-Wegener-Institute Bremerhaven

Ellen Weihe
Alfred-Wegener-Institute Bremerhaven

Magnus Lucassen
Alfred-Wegener-Institute Bremerhaven

Research areas

King George Island (South 62°, West 58°) and surroundings

Publications

Obermüller B, Karsten U, Pörtner HO, Abele D, 2003. Effects of UV-radiation on oxidative stress parameters and uptake of mycosporine-like amino acids (MAAs) in polar marine amphipods. In: Antarctic Biology in a Global Context. (Eds. Huiskes AHL, Gieskes WWC, Rozema RML, Schorno SM, Vies SM, Wolff WJ), Backhuys Publishers, Leiden, The Netherlands, 63-68.

Abele D, Puntarulo S, 2004. Formation of reactive species and induction of antioxidant defense systems in polar and temperate marine invertebrates and fish. Review, CBP 138A (4): 405-415.

Obermüller B, Karsten U, Abele D, 2005. Response of oxidative stress parameters and sunscreening compounds during experimental exposure to maximal natural UVB-radiation in Arctic amphipods. J. Exp. Mar. Biol. Ecol. 323, 100-117.

Obermüller B, Puntarulo S, Abele D, 2007. UV-tolerance and instantaneous physiological stress responses of two Antarctic amphipod species Gondogeneia antarctica and Djerboa furcipes during exposure to UV radiation. Marine Environmental Research. 64, 267–285.

Weihe E, Abele D, 2008. Differences in the physiological response of inter- and subtidal Antarctic limpets (/Nacella concinna/) to aerial exposure. Aquatic Biology 4, 155–166.

Weihe E, Kriews M, Abele D, 2009. Differences in heavy metal concentrations and in the response of the antioxidant system to hypoxia and air exposure in the Antarctic limpet Nacella concinna. Marine Environmental Research (Accepted).

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Research funding organisation

German Research Foundation

Project numbers: DA 124/7
Funding period: October 2005 - December 2008