Molecular events in the evolution of freeze resistant fish: type I AFP in cunner (Tautogolabrus adspersus) and type III AFP in the rock gunnel (Pholis gunnellus) and radiated shanny (Ulvaria subbifurcata)

Hobbs, Rod S. (2014) Molecular events in the evolution of freeze resistant fish: type I AFP in cunner (Tautogolabrus adspersus) and type III AFP in the rock gunnel (Pholis gunnellus) and radiated shanny (Ulvaria subbifurcata). Doctoral (PhD) thesis, Memorial University of Newfoundland.

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Abstract

Type I Antifreeze proteins (AFPs) have been fully characterized in two teleost Orders; Pleuronectiformes and Scorpaeniformes. Results from this study have confirmed that cunner [Tautogolabrus adspersus (Family Labridae)], a member of a third Order (Order Perciformes), also produce a type I AFP. This finding solidifies the hypothesis that type I AFPs are multiphyletic in origin and suggests that they represent remarkable examples of convergent evolution within three Orders of teleost fish. The cunner AFP was purified from blood plasma and found to belong to what is now known as classical type I AFP with their small size (mass = 4095.16 Da), alanine-richness (>57 mol%), high α-helicity (>99%) with the ability to undergo reversible thermal denaturation, 11-amino acid (ThrX10) repeat regions within the primary structure, the capacity to impart a hexagonal bipyramidal shaping to ice crystals and the conservation of an ice binding site found in many of the other type I AFPs. cDNA library sequencing indicated that the protein is produced without a signal sequence yet the translated product of the AFP cDNA suggests that it codes for the AFP isolated from plasma. Despite the fact that cunner produce AFPs, the AFP concentration within the blood is too low to provide whole body freeze protection. Southern and northern blot analyses carried out in this study establishes that the cunner AFP genes belong to a multigene family that is predominantly expressed in external epithelia (skin and gill filaments). These results support the hypothesis that the survival of cunner in icy waters is attributable in part to epithelial AFPs that help block ice propagation into their interior milieu. In contrast to the cunner, heterospecifics occupying the same habitat have greater freeze protection because they produce AFPs in the liver for export to the plasma as well as in external epithelia. Since the external epithelia would be the first tissue to come into contact with ice it is possible that one of the earliest steps involved in the evolution of freeze resistant fish could have been the expression of AFPs in tissues such as the skin. I suggest that this epithelial-dominant AFP expression represents a primitive stage in AFP evolution and propose that cunner began to inhabit “freeze-risk ecozones” more recently than heterospecifics. Type III AFPs have been identified in a single suborder of fish (Zoarcoidei). In the eelpouts (family Zoarcoidae) and wolffishes (family Anarhichadidae), freeze protection is accomplished through the cooperation of two distinct groups of AFP isoforms: SP-Sephadex (SP) and QAE-Sephadex (QAE) binding types. Although members from most families within the suborder Zoarcoidei are known to produce type III AFP, the cooperative action between AFP isoforms has not been confirmed outside of these two families. Through sequence analysis of AFP cDNAs isolated in this study, I have confirmed the presence of both SP and QAE type III isoforms in two additional families within this suborder: Pholidae [rock gunnel (Pholis gunnellus)] and Stichaeidae [radiated shanny (Ulvaria subbifurcata)]. These results support the hypothesis that cooperativity between SP and QAE isoforms evolved prior to the divergence of families within the suborder Zoarcoidei.

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