Clive W. Evans

Nonhepatic origin of notothenioid antifreeze reveals pancreatic synthesis as common mechanism in polar fish freezing avoidance

Phylogenetically diverse polar and subpolar marine teleost fishes have evolved antifreeze proteins (AFPs) or antifreeze glycoproteins (AFGPs) to avoid inoculative freezing by internalized ice. For over three decades since the first fish antifreeze (AF) protein was discovered, many studies of teleost freezing avoidance showed hepatic AF synthesis and distribution within the circulation as pivotal in preventing the blood, and therefore the fish, from freezing. We have uncovered an important twist to this long-held paradigm: the complete absence of liver synthesis of AFGPs in any life stage of the Antarctic notothenioids, indicating that the liver plays no role in the freezing avoidance in these fishes. Instead, we found the exocrine pancreas to be the major site of AFGP synthesis and secretion in all life stages, and that pancreatic AFGPs enter the intestinal lumen via the pancreatic duct to prevent ingested ice from nucleating the hyposmotic intestinal fluids. AFGPs appear to remain undegraded in the intestinal milieu, and the composition and relative abundance of intestinal AFGP isoforms are nearly identical to serum AFGPs. Thus, the reabsorption of intact pancreas-derived intestinal AFGPs, and not the liver, is the likely source of circulatory AFGPs in notothenioid fishes. We examined diverse northern fish taxa and Antarctic eelpouts with hepatic synthesis of bloodborne AF and found that they also express secreted pancreatic AF of their respective types. The evolutionary convergence of this functional physiology underscores the hitherto largely unrecognized importance of intestinal freezing prevention in polar teleost freezing avoidance, especially in the chronically icy Antarctic waters.

Induction of cytochrome P4501A (CYP1A) in Trematomus bernacchii as an indicator of environmental pollution in Antarctica: assessment by quantitative RT-PCR

Abstract Although most of Antarctica is relatively pristine, high levels of pollutants have been recorded in localised areas such as in the vicinity of scientific bases like McMurdo Station. We have used the Antarctic fish Trematomus bernacchii as an indicator species to assess the level of impact of these pollutants on the local biota. Fish were collected from Winter Quarters Bay (adjacent to McMurdo Station) and Backdoor Bay (remote from human activities). Liver samples from individual fish were used in the preparation of total RNA from which the level of expression of the cytochrome P4501A gene ( CYP1A ), known to be responsive to polycyclic aromatic hydrocarbons (PAHs), was determined by quantitative competitive reverse-transcriptase polymerase chain reaction (RT-PCR). Samples of bile from the same fish were analysed for fluorescent aromatic compounds (FACs) at naphthalene and phenanthrene wavelengths to provide an indication of exposure to organic compounds such as the PAHs. The levels of biliary FACs in fish from Winter Quarters Bay were approximately 2-fold higher than those in fish from Backdoor Bay, whereas there was an average 37-fold increase in CYP1A expression between fish taken from the two sites. The extent of CYP1A induction correlated positively with biliary FACs levels, indicating the potential of quantitative competitive RT-PCR as a sensitive molecular approach to pollution impact assessment. Fish from Winter Quarters Bay also had significantly higher hepatosomatic and gonadosomatic indices indicative of altered organ function, although the extent to which this might be related to pollution is uncertain.

Freezing resistance of antifreeze-deficient larval Antarctic fish

SUMMARY Antarctic notothenioids, along with many other polar marine fishes, have evolved biological antifreeze proteins (AFPs) to survive in their icy environments. The larvae of Antarctic notothenioid fish hatch into the same frigid environment inhabited by the adults, suggesting that they must also be protected by sufficient AFPs, but this has never been verified. We have determined the contribution of AFPs to the freezing resistance of the larvae of three species: Gymnodraco acuticeps, Pagothenia borchgrevinki and Pleuragramma antarcticum. Of the three, only P. borchgrevinki larvae are protected by high, adult levels of AFPs. Hatchling G. acuticeps and P. antarcticum have drastically inadequate AFP concentrations to avoid freezing at the ambient seawater temperature (-1.91°C). We raised G. acuticeps larvae and measured the AFP levels in their blood for ∼5 months post hatching. Larval serum freezing point was -1.34±0.04°C at the time of hatch; it began to decrease only after 30 days post hatch (d.p.h.), and finally reached the adult value (-2.61±0.03°C) by 147 d.p.h. Additionally, AFP concentrations in their intestinal fluids were very low at hatching, and did not increase with age throughout a sampling period of 84 d.p.h. Surviving in a freezing environment without adequate AFP protection suggests that other mechanisms of larval freezing resistance exist. Accordingly, we found that G. acuticeps hatchlings survived to -3.6±0.1°C while in contact with external ice, but only survived to -1.5±0.0°C when ice was artificially introduced into their tissues. P. antarcticum larvae were similarly resistant to organismal freezing. The gills of all three species were found to be underdeveloped at the time of hatch, minimizing the risk of ice introduction through these delicate structures. Thus, an intact integument, underdeveloped gill structures and other physical barriers to ice propagation may contribute significantly to the freezing resistance and survival of these larval fishes in the icy conditions of the Southern Ocean.

Synthesis and antifreeze activity of fish antifreeze glycoproteins and their analogues

Fishes from both Arctic and Antarctic waters produce antifreeze glycoproteins (AFGPs) that modify and inhibit the growth of ice crystals, allowing them to survive in extreme cold conditions. These glycoproteins exhibit thermal hysteresis activity, i.e. they work in a non-colligative manner, separating the melting and freezing points of a solution. Such compounds have many potential applications; unfortunately their development is hampered by the difficulty of obtaining pure material. The synthesis of AFGPs is therefore a challenge that numerous groups have been tackling. The AFGPs consist predominantly of a repetitive three amino acid unit (Ala-Ala-Thr)n with the disaccharide β-D-galactosyl-(1–3)-α-D-N-acetylgalactosamine attached to the hydroxyl oxygen of each threonine residue. A large number of analogues have also been synthesized in order to find compounds that exhibit the same activity but that are easier to prepare. Starting from the early years of the AFGP discovery and including the more recent research, this perspective summarizes the different routes used to synthesize native AFGPs and lists the most relevant analogues synthesized, along with some information on their synthesis and their antifreeze activity, if evaluated. In this perspective we have taken special care to differentiate compounds that induce thermal hysteresis, compounds that modify the normal growth habit of ice crystals, and compounds that exhibit recrystallization inhibition properties.

Cell adhesion and metastasis

There is no single phenotypic trait whose exclusive expression correlates universally with metastatic behaviour. Nevertheless, several lines of evidence point towards adhesive phenomena as mediating some crucial steps in the malignant spread of particular tumour types. These steps include detachment from the primary, invasion of the ECM including include detachment from the primary, invasion of the ECM including structures such as the basement membrane and organ capsules, and lodgement in the vessels of remote organs. Furthermore, there is evidence that adhesive phenomena can contribute at least in part to the selective patterns of spread shown by a variety of tumour cell types. The molecular dissection of the adhesive events involved in these processes has only just begun. It is clear already, however, that several different determinants are likely to be involved and the clinical application of this knowledge is unlikely to be immediate.

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

Significance Antarctic notothenioid fishes are protected from freezing by antifreeze proteins (AFPs) that bind to invading ice crystals and inhibit their growth. Paradoxically, accumulation of AFP-stabilized ice could be lethal. Whether and how fishes eliminate internal ice is unknown; one hypothesis is that it melts during summer warming episodes. However, prior in vitro evidence indicates that AFPs also inhibit melting. Our study establishes that pronounced melting inhibition occurs in vivo (i.e., superheated ice occurs inside notothenioid fishes). Our long-term temperature record of a high-latitude Antarctic fish habitat indicates that summer warming does not overcome AFP-induced superheating to reliably rid fishes of ice. Evolution of the life-saving AFPs exacts a cost: the risk of lifelong accumulation of damaging internal ice crystals. 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.

Spawning behaviour and early development in the naked dragonfish Gymnodraco acuticeps

Nesting sites of the naked dragonfish Gymnodraco acuticeps have been identified in 15–35 m water under fast ice adjacent to McMurdo Station, making it possible to examine embryonic development and early larval growth. Egg-laying (predominantly in October) is preceded by a distinctive whirling behavioural pattern driven by the male prodding the side of the females abdomen. The eggs (3.42 ± 0.19 mm in diameter) are laid on rocks as a single adherent layer (c. 2500 per patch). Development is unusually protracted, the first cleavage occurring after about 24 hr at about −1.9°C. Hatching occurs about 10 months post-fertilization, beginning soon after the sun rises above the horizon. During this period one of the parents may act as a guard in an attempt to keep predators at bay. Upon hatching, the larvae (12.09 ± 0.36 mm long) swim towards the surface ice where they presumably seek refuge. Yolk absorption is complete in about 15 days. Larvae (grown in aquaria at a density of 0.7 larvae l−1) display an average daily growth rate of 0.42% over nine weeks. Hatching in aquaria can occur up to 100 days in advance of that seen in the field, suggesting that under natural conditions hatching may be delayed until an appropriate stimulus (such as the return of the sun) is received.

Trapping and imaging of micron‐sized embryos using dielectrophoresis

Development of dielectrophoretic (DEP) arrays for real‐time imaging of embryonic organisms is described. Microelectrode arrays were used for trapping both embryonated eggs and larval stages of Antarctic nematode Panagrolaimus davidi. Ellipsoid single‐shell model was also applied to study the interactions between DEP fields and developing multicellular organisms. This work provides proof‐of‐concept application of chip‐based technologies for the analysis of individual embryos trapped under DEP force.

Molecular portrait of lens gap junction protein MP70

A 70-kDa membrane protein (MP70) is a component of the lens fiber gap junctions. Its membrane topology and its N-terminal sequence are similar to those of the connexin family of proteins. Some features of MP70 containing fiber gap junctions are, however, distinct from gap junctions in other mammalian tissues: (i) Lens connexons form crystalline arrays only after cleavage of junctional proteins in vitro. These hexagonal arrays have a periodicity of 13.6 nm which is significantly larger than the 8- 9-nm spacing of liver and heart gap junctions. (ii) Lens fiber gap junctions dissociate in low concentrations of nonionic detergent and this provides an avenue to purify MP70 directly from a membrane mixture. Isolated MP70 in the form of 17 S structures has an appearance consistent with connexon pairs. (iii) The C-terminal half of MP70 is cleaved in situ by a lens endogenous calcium-dependent protease. The processed from MP38 remains in the membrane and is abundant in the central region of the lens. A testable hypothesis for MP70 function is presented.

How do Antarctic notothenioid fishes cope with internal ice? A novel function for antifreeze glycoproteins

Abstract Antarctic fishes survive freezing through the secretion of antifreeze glycoproteins (AFGPs), which bind to ice crystals to inhibit their growth. This mode of action implies that ice crystals must be present internally for AFGPs to function. The entry and internal accumulation of ice is likely to be lethal, however, so how do fishes survive in its presence? We propose a novel function for the interaction between internal ice and AFGPs, namely the promotion of ice uptake by splenic phagocytes. We show here that i) external mucus of Antarctic notothenioids contains AFGPs and thus has a potential protective role against ice entry, ii) AFGPs are distributed widely through the extracellular space ensuring that they are likely to come into immediate contact with ice that penetrates their protective barriers, and iii) using AFGP-coated nanoparticles as a proxy for AFGP adsorbed onto ice, we suggest that internal ice crystals are removed from the circulation through phagocytosis, primarily in the spleen. We argue that intracellular sequestration in the spleen minimizes the risks associated with circulating ice and enables the fish to store the ice until it can be dealt with at a later date, possibly by melting during a seasonal warming event.