Antifreeze protein-induced superheating of ice inside Antarctic notothenioid fishes inhibits melting during summer warming

Abstract

Antifreeze proteins (AFPs) of polar marine teleost fishes are widely recognized as an evolutionary innovation of vast adaptive value in that, by adsorbing to and inhibiting the growth of internalized environmental ice crystals, they prevent death by inoculative freezing. Paradoxically, systemic accumulation of AFP-stabilized ice could also be lethal. Whether or how fishes eliminate internal ice is unknown. To investigate if ice inside high-latitude Antarctic notothenioid fishes could melt seasonally, we measured its melting point and obtained a decadal temperature record from a shallow benthic fish habitat in McMurdo Sound, Antarctica. We found that AFP-stabilized ice resists melting at temperatures above the expected equilibrium freezing/melting point (eqFMP), both in vitro and in vivo. Superheated ice was directly observed in notothenioid serum samples and in solutions of purified AFPs, and ice was found to persist inside live fishes at temperatures more than 1 °C above their eqFMP for at least 24 h, and at a lower temperature for at least several days. Field experiments confirmed that superheated ice occurs naturally inside wild fishes. Over the long-term record (1999-2012), seawater temperature surpassed the fish eqFMP in most summers, but never exceeded the highest temperature at which ice persisted inside experimental fishes. Thus, because of the effects of AFP-induced melting inhibition, summer warming may not reliably eliminate internal ice. Our results expose a potentially antagonistic pleiotropic effect of AFPs: beneficial freezing avoidance is accompanied by melting inhibition that may contribute to lifelong accumulation of detrimental internal ice crystals.

Keywords: McMurdo Sound temperature; antagonistic pleiotropy; antifreeze glycoprotein; antifreeze potentiating protein; melting hysteresis.

Fig. 1.

Locations of study sites in McMurdo Sound, Antarctica. Colored circles indicate approximate locations of conductivity (salinity), temperature, and depth (CTD) profiler casts, field experiments, collection sites, and a long-term temperature record. McMurdo Sound is covered by sea ice for much of each year. Dashed lines indicate the annual minimum extent of land-fast sea ice in 2 y of the study (estimated from satellite imagery). GPS coordinates are given in Table S3.

Fig. 2.

Superheating of ice in fish serum is caused by AFPs. (A) The eqFMPs of the blood sera of various notothenioid species (Upper) and of other fluids of T. pennellii (Lower) were determined with a vapor pressure osmometer (mean ± SE; n = 3–15 individuals for each point). The mean serum eqFMP for all species tested (−1.04 °C) is indicated by a dashed line; we used this value as a proxy for the eqFMP of McMurdo Sound notothenioid fishes. EDF, extradural fluid; IF, intestinal fluid; and VH, vitreous humor. Species names shortened from T. pennellii, T. bernacchii, T. hansoni, P. borchgrevinki, and D. mawsoni. (B) Ice persisted in vitro at temperatures both above and below solution eqFMPs. Nonequilibrium hFPs (shaded bars) and hMPs (white bars) shown as the difference from the eqFMPs of serum (Upper) and of purified AFPs at physiological concentration in buffer (Lower). Values (mean ± SE) for a single sample assayed three to four times. The greatest observed values of MH and FH are enumerated, with the corresponding MH:FH ratio shown at right.

Fig. 3.

Superheated ice occurs inside live notothenioid fishes. (A) Fraction of McMurdo Sound notothenioid fishes harboring experimentally introduced (circles) or naturally acquired (crosses) internal ice after a 24-h experimental warming treatment at the indicated aquarium temperature (mean with range error bars). Peak annual seawater temperatures for the McMurdo site are indicated by arrowheads and labeled by oceanographic year (Table S1). The fish eqFMP (−1.04 °C) and the maximum temperature ever recorded at the McMurdo site (−0.10 °C) are shown by dashed and dotted lines, respectively. (B) Fraction of T. pennellii harboring internal ice as a function of time at −0.73 °C (mean, range −0.82 to −0.65 °C), 0.31 °C above their eqFMP. In both experiments, aquarium temperature was measured within 30 cm of the fishes every 2 s with a high-resolution logger. n = 8–15 individuals for each datapoint.

Fig. 4.

Superheated ice occurs in nature. (A) Profiles of the water column, from underneath surface ice to the sea floor, at collection sites near Cape Evans, Ross Island in January 2013 revealed seawater temperatures above the T. bernacchii eqFMP (mean −1.05 °C, dashed line). (B) Nevertheless, internal ice persisted in a large fraction of individuals collected from the bottom at these sites. Control experiments verified that fishes did not acquire internal ice during the brief transit through the colder surface layer during capture.

Fig. 5.

Superheating further restricts infrequent opportunities for melting internal ice. (A) Temperature record for the McMurdo site. Peak annual temperatures are enumerated. Seawater temperature occasionally exceeded the fish eqFMP (lower dashed line). However, in over a decade of logging at this shallow benthic (25–40 m) fish habitat, it never exceeded the highest temperature to which internal ice persisted in vivo under laboratory conditions (+0.08 °C, upper dashed line). Experimental results from this study strongly support the inference that superheated internal ice could persist under the thermal conditions indicated in the shaded areas throughout the figure. (B) Vertical seawater temperature profile (solid line) representative of winter conditions. Seawater in the upper 30 m is below the pressure- and salinity-dependent in situ freezing point (dashed line), resulting in a high probability of shallow-living fishes contacting and internalizing abundant environmental ice. (C–E) Period of peak annual temperatures shown for years with (C) the longest continuous period above the fish eqFMP (9.79 d in 2000), (D) the greatest cumulative time above the eqFMP (18.76 d in 2009), and (E) the highest temperature (−0.10 °C in 2012). (F) Summertime seawater temperature profiles from multiple sites in McMurdo Sound. Temperatures above the fish eqFMP occur only in the top 100 m of the water column, deeper waters are perennially frigid. Profile sites: January 11, mid-Sound (yellow); January 17, Cape Evans (pink), Dailey Islands (gray), Granite Harbor (orange), McMurdo (green), New Harbor (blue); February 12, mid-Sound (black).