Dr Clive W Evans

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Honorary, Academic

Research | Current

Fish Antifreeze

Developmentally regulated expression of antifreeze in Antarctic fish

crystalThe Southern Ocean around the coast of Antarctica is a thermally stable environment in which the sea water temperature hovers close to its freezing point around -1.93C. Most marine teleosts have an equilibrium freezing point of about -0.7C, substantially higher than that of sea water, and they therefore cannot survive by supercooling in the icy Antarctic waters (DeVries et al., 1988). Notothenioid fishes thrive in this freezing environment, however, due to their capacity to produce antifreeze glycoproteins (AFGPs). AFGPs bind to and inhibit the growth of minute ice crystals that occasionally enter the fish, thus preventing their body fluids from freezing (DeVries and Cheng, 1992). They are a key evolutionary innovation that conferred freeze avoidance and empowered the adaptive radiation of the notothenioids in the poorly colonised, frigid waters of the Southern Ocean some 5-15 million years ago (Cheng et al., 1997; Eastman and McCune, 2000).

The site of AFGP synthesis in notothenioids is unknown

Trematomus bernacchiiAs many as 20 AFGP isoforms, all containing the same glycotripeptide, are synthesized in notothenioid species (Chen et al., 1997; Hsiao et al., 1990). The AFGPs evolved from a pancreatic trypsinogen-like protease (Chen et al., 1990; Cheng and Chen, 1990) and the isoforms are encoded by a large multigene family (Cheng, 1996). While the molecular basis for AFGP production is well understood, the site of AFGP synthesis remains an unresolved question. Unlike most other antifreeze-bearing fishes where the liver is the major synthetic site, adult notothenioids show only minimal AFGP transcription in this organ. Nothing is known about AFGP production in notothenioid larvae, nor when and where AFGPs are first synthesised during their embryogenesis.

The naked dragonfish Gymnodraco acuticepsIn joint work with Art DeVries and Chris Cheng from the University of Illinois at Urbana-Champaign, we are currently investigating the sites of tissue synthesis of AFGPs in larval and adult dragonfish (Gymnodraco acuticeps), and the tempo of their upregulation during development. We are in an unique position to undertake these studies because we have ready access to larval, adult and fecund dragonfish in McMurdo Sound (Antarctica).

In preliminary work we have successfully fertilized mature oocytes in vitro, and maintained them throughout embryogenesis. We have also found that dragonfish eggs are laid in freezing seawater in early October. Although they are hypo-osmotic to seawater the eggs do not freeze, suggesting fortification with maternal antifreeze. Newly-hatched larvae swim directly to the surface-ice platelet layer to avoid predators, indicating that they contain AFGP and that a switch to zygotic production must take place during development.

The sponge cemetery off Bratina Island, McMurdo Sound, AntarcticaWe are using a combination of molecular biology (quantitative RT-PCR to enumerate transcript levels), immunocytochemistry (to localise the sites of AFGP expression and/or storage), and biochemistry (western blotting to identify specific AFGP isoforms) to analyse AFGP synthesis during development. Eggs at different times (before and after fertilisation) are being analysed in parallel for AFGP activity in the perivitelline, yolk and embryonic compartments using differential osmometry to estimate antifreeze activity.

Unique primers designed from dragonfish AFGP cDNAs will then be used in quantitative RT-PCR assays and specific AFGP antibodies will be employed for western blotting and immunocytochemistry in this combinatorial approach to determine the sites and tempo of AFGP synthesis during development.

Surviving the freezing waters of Antarctica demands tightly regulated control of AFGP production at all life stages because its synthesis requires a substantial energy investment. Our studies will open new pathways for gaining insight into how this control is achieved at the molecular and cellular levels. They will also generate information on the rates of larval development and growth relevant to Antarctic fisheries management in which New Zealand has increasing involvement.

Research Project Availability

A number of different research projects focussing on Antarctica are available in the Evans laboratory. Please contact Clive Evans if you wish to discuss the possibilities of Antarctic research further and receive more specific details.

References

  • Chen, L, DeVries, AL and Cheng, C-HC (1997): Evolution of antifreeze glycoprotein gene from a trypsinogen gene in Antarctic notothenioid fish. Proc Natl Acad Sci 94: 3811-3816.
  • Cheng, C-HC (1996): Genomic basis for antifreeze glycopeptide heterogeneity and abundance in Antarctic notothenioid fishes. In: Gene Expression and Manipulation in Aquatic Organisms, Soc. of Exp. Biol. Seminar Series 58 (Eds. S. Ennion & G. Goldspink), Cambridge University Press, pp. 1-20.
  • Cheng, C-HC and Chen, L (1999): Evolution of an antifreeze glycoprotein. Nature 40: 443-444. DeVries, AL (1988): The role of antifreeze glycopeptides and peptides in the freezing avoidance of Antarctic fishes. Comp Biochem Physiol 90B: 611-621.
  • DeVries, AL and Cheng, C-HC (1992): The role of antifreeze glycopeptides and peptides in the survival of cold water fishes. In: Water and Life: Comparative Analysis of Water Relationships at the Organismic, Cellular, and Molecular Levels (Eds. G.N. Somero, C.B. Osmond, C.L. Bolis), Springer Verlag, pp. 303-315.
  • Eastman, JT and McCune, AR (2000): Fishes on the Antarctic continental shelf: evolution of a marine species flock?. J Fish Biol 57: 84-102.
  • Hsiao, K-C, Cheng, C-HC, Fernandes, IE, Detrich, HW and DeVries, AL (1990): An antifreeze glycopeptide gene from the Antarctic cod Notothenia coriiceps neglecta encodes a polyprotein of high peptide copy number. Proc Natl Acad Sci 87: 9265-9269


Molecular Ecotoxicology and Disease

There are a number of different research strategies available to assess biological impact at the molecular level although all have the same common goals: to provide a measure of the state of the environment though its impact on the biota and, where appropriate, to use the information obtained as a reference database for recommending any necessary environmental improvement. Our basic approach has been to use the extraordinary sensitivity of RT-PCR amplification as a tool in the quantitative assessment of the effects on fish of exposure to environmental pollutants and to use this as one measure of environmental impact (Evans et al., 2000, 2001; Miller et al., 1999).

mt_v_cu Correlation between metallothionein induction as determined by quantitative competitive (qc) RT-PCR and hepatic copper levels in the yellowbelly flounder Rhombosolea leporina collected near Auckland, New Zealand.

In our initial studies this was achieved by measuring the induction of specific genes such as those for metallothionein (which reflects heavy metal exposure) and cytochrome P4501A (which reflects exposure to specific hydrocarbons). In order to assess the appropriateness of the RT-PCR approach, correlates have been sought between our molecular biological studies and levels of specific pollutants in fish tissues as well as with a spectrum of fish health indicators.

wqbfish Stomach contents of Trematomus bernacchii collected near the sewage outfall at McMurdo Station, Antarctica. Peas, corn and carrot are visible in the opened stomach.

Molecular ecotoxicology offers a powerful approach which supplements the more classic procedures of environmental toxicology. Because it provides information at the earliest response level to exposure it has the capacity to serve as an early warning indicator of potentially more significant and widespread effects. Molecular ecotoxicology is thus a key parameter in monitoring the environment and recommending steps for any necessary improvement. Additionally, since knowledge of the molecular and cellular responses to particular pollutants also contributes to our understanding of the aetiology of specific bioeffects, the molecular ecotoxicology approach provides a route to the development of treatments for improving organismal health.

xcell X-cell disease in the gills of Trematomus bernacchii collected from Winter Quarters Bay, Antarctica. The X cells are visible as large cells with distinct nucleoli in the basal region of the lamellae. Thrombus-like inclusions are obvious within aneurysms of the fused secondary gill lamaellae.

Research Project Availability

A number of different research projects focussing on Molecular Ecotoxicology are available in the Evans laboratory. Please contact us if you wish to receive more specific details.

Selected publications and creative works (Research Outputs)