In order to investigate how Antarctic fish deal with ice in their blood circulatory system, Trematomus bernacchii and Pagothenia borchgrevinki Antarctic fish were captured for experimental investigation. A variety of biochemical techniques were used to isolate Anti-Freeze Glyco-Proteins (AFGPs) from Antarctic fish blood, which was then conjugated directly to a fluorescent marker. We synthesised ... silicon dioxide nanobeads incorporating a fluorescent probe and coupled these to AFGP via a multimeric linking protein or dendrimer based on G4.5 poly(amidoamine). These modified nanobeads, consisting of a fluorescent particle surrounded by AFGPs, were designed to serve as a proxy for ice crystals that have bound AFGPs. When introduced into the circulation of the fish, we were able to follow the fate of free fluorescent AFGP and particle-bound fluorescent AFGP (the latter serving as a proxy for ice in the circulation). Frozen and alcohol-fixed tissues were returned to New Zealand for further laboratory analysis.
Previous evidence from our group has suggested that the spleen is the only location where trapping of ice crystals takes place, and thus we targeted this organ in our studies. We are also interested in determining how high levels of AFGPs are maintained in the blood, since most of it seems to be secreted into the gut where it would be expected to be lost with the faeces.
We also sampled a variety of Antarctic fishes to determine where AFGPs are synthesised and the nature of their ultimate fate using fluorescent microscopy with specific antibodies we had prepared earlier. Finally, we synthesised 15N-labelled AFGP8 and fed it to fish to determine how AFGPs are taken up from the gut and into the blood.