We identified two ice-binding protein (IBP) sequences, named EFsymbAFP and EFsymbIBP, from a putative bacterial symbiont of the Antarctic psychrophilic ciliate Euplotes focardii. EFsymbAFP is 57.43% identical to the antifreeze protein (AFP) from the Stigmatella aurantiaca strain DW4/3-1, which was isolated from the Victoria Valley lower glacier. EFsymbIBP is 53.38% identical to the IBP from the Flavobacteriaceae bacterium strain 3519-10, isolated from the glacial ice of Lake Vostok. EFsymbAFP and EFsymbIBP are 31.73% identical at the amino acid level and are organized in tandem on the bacterial chromosome. The relatively low sequence identity and the tandem organization, which appears unique to this symbiont, suggest an occurrence of horizontal gene transfer (HGT). Structurally, EFsymbAFP and EFsymbIBP are similar to the AFPs from the snow mould fungus Typhula ishikariensis and from the Arctic yeast Leucosporidium sp. AY30. A phylogenetic analysis showed that EFsymbAFP and EFsymbIBP cluster principally with the IBP sequences from other Antarctic bacteria, supporting the view that these sequences belong to an Antarctic symbiontic bacterium of E. focardii. These results confirm that IBPs have a complex evolutionary history, which includes HGT events, most probably due to the demands of the environment and the need for rapid adaptation.

The Antarctic psychrophilic ciliate Euplotes focardii manifests a dramatic reduction in the activation of its hsp70 gene in response to a heat-shock, while oxidative and chemical stresses activate the transcription of this gene to appreciable extents. To investigate the genetic causes of this eccentric behaviour of E. focardii in the hsp70 gene transcription activation, we carried out a comparative structural analysis of this gene between E. focardii and another Antarctic Euplotes, E. nobilii, which manifests a psychrotrophic behaviour and an inducible thermal response. No substantial difference was detected in the organization of the hsp70 5' promoter region, both species bearing canonical regulatory cis-acting elements deputed to bind transcriptional trans-activating factors. Adenine-rich elements favouring mRNA degradation were instead detected in the hsp70 3' regulatory region of E. nobilii, but not in that of E. focardii. These observations lend further support to the hypothesis that the causes of the Euplotes focardii unresponsiveness to thermal stress resides in some structural, or functional modifications of transcriptional trans-activating factors.

Following the break-up of Gondwana, the drift of Antarctica to its present position and the establishment of the Antarctic Convergence, fish evolution was characterized by adaptation to progressive cooling of the environment. The decrease of erythrocyte number and haemoglobin concentration in the blood of Antarctic teleosts raises several questions concerning the physiology of respiration and the enzymatic role of erythrocytes. Our study of the molecular basis of cold adaptation includes the relationship between molecular structure and biological function of haemoglobins. Species of the suborder Notothenioidei, largely confined within the Convergence, have only one major haemoglobin, which displays the Root effect in oxygen binding; on the other hand, Zoarcidae (a family found at all latitudes) have four or five haemoglobins, only one of which displays the Root effect. In addition, our data indicate that the physiological relevance of erythrocyte-like cells, present in very small number in the blood of haemoglobinless Channichthyidae, may be linked to higher content of enzymes, such as glucose-6-phosphate dehydrogenase, in comparison with erythrocytes of red-blooded fishes.