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Adaptation of Poikilotherms to Subzero Temperatures

• Adaptation mechanisms previously discussed are reinforced with additional capabilities to prevent injuries that can occur due to ice formation.

Freeze Damage

• Freeze damage is predominantly caused by cell dehydration.

• Key Points:

  • Most organisms cannot endure freezing conditions.

  • Intracellular ice formation is lethal to cells.

  • Most ice formation occurs extracellularly, leading to problems such as:

  • Dehydration of cellular water.

  • Changes in osmotic balance due to movement of extracellular fluids.

Cellular and Extracellular Dynamics

• Diagrammatic representation includes:

  • Extracellular ice formation.

  • Structural components of a cell, including the nucleus, cytosol, and osmotic processes involved.

• Osmosis:

  • Ice crystals forming in the slowly cooled extracellular fluid exclude solutes.

  • This increases solute concentration in the unfrozen extracellular fluid, prompting osmotic water loss from cells.

  • Osmotic changes reduce intracellular water, elevating solute concentration in shrunken cells, thereby lowering the freezing point of the cytosol.

Cell Dehydration Effects

• Dehydration leads to several negative outcomes in cells:

  • Reduced water content inside cells (Dewatering process).

  • Decreased cell volume leading to solute precipitation.

  • Potential rupture of cellular membranes.

  • Denaturation of proteins and altered protein interactions.

Mechanisms to Escape Injury from Subzero Temperatures

1. Behavioral Avoidance

2. Rapid Cold Hardening

   • Method of avoiding cold shock.

3. Cold Acclimatization

   • A) For freeze-susceptible species: Increase supercooling ability and avoid forming ice.

   • B) For freeze-tolerant species: Allowing some ice formation within limits.

4. Developmental Preparedness

Freezing Resistance in Animals

• The freezing resilience of animals differs based on their blood osmolarity relative to their environment:

  • Isoosmotic to seawater organisms generally do not freeze, unless all surrounding water freezes.

  • Hyperosmotic organisms to freshwater will not freeze.

  • Saltwater teleost fish are hypo-osmotic to seawater, which renders their freezing points above seawater, presenting a freezing challenge.

  • Terrestrial organisms facing subzero temperatures may be susceptible to freezing.

Marine Teleost Fish Adaptation

• Supercooling:

  • Certain marine teleosts can supercool to -1.9°C without coming into contact with ice.

  • Behaviorally, they migrate to deeper waters to avoid ice contacts.

• Ice Fish behavior:

  • Fish immobilized near ice risk freezing.

  • Fish in deep water are less likely to freeze.

Freezing Point Depression in Marine Teleosts

• Some marine teleost fish can depress their freezing point below that of seawater.

  • Achieving this isoosmotic state is challenging.

  • Enzymatic functions and renal constraints emerge as problems.

  • These fish do not increase glucose or glycerol levels significantly but utilize Antifreeze Proteins (AFPs) to achieve freezing point reduction.

Antifreeze Proteins (AFPs)

• First identified were Antifreeze Glycoproteins (AFGPs).

• Structure:

  • Comprised of repeating tri-peptides with attached carbohydrate groups.

  • Eight distinct AFGPs are present within species, varying in repeat units.

  • Example Structure:

  • AFGP structure: Alanine - Alanine - Threonine with N-acetyl galactosamine or galactose.

Mechanism of Antifreeze Proteins

• AFPs do not lower the freezing point through colligative means, distinguishing them from other low molecular weight antifreezes.

• Mode of action involves direct binding to ice, which hinders growth of ice crystals.

• Production of Thermal Hysteresis is a key characteristic:

  • Difference between melting point and freezing point, resulting in specific temperature ranges where ice and water cohabitate (e.g., $(MP = -0.9°C; FP = -2.5°C; TH = 1.4°C)$).

Types of Antifreeze Proteins (AFPs)

1. AFGPs (e.g., Antarctic cods)

2. Type 1 AFP (winter flounder) - Alanine-rich, alpha-helix structure.

3. Type 2 AFPs (sea ravens) - Cysteine-rich.

4. Type 3 AFPs (eel pouts) - Non-alanine, non-cysteine-rich.

5. Type 4 AFPs (long-horned sculpin) - Glycine-rich.

Gene and Protein Study in Antifreeze Production

• Numerous AFP genes have been cloned, leading to findings of multigene families across fish types.

• Ongoing research involves structure/function assessments of recombinant AFP molecules to determine ice binding efficiency and growth inhibition features.

• Potential commercial applications are being explored.

Seasonal Variations in Antifreeze Production

• A graphical representation (Figure 10.25) illustrates seasonal changes in antifreeze protection in winter flounder, indicating fluctuations in protein concentration and freezing points across different months.

Regulation of Antifreeze Proteins (AFPs)

• Seasonal regulation of AFP involves:

  • Decrease in Insulin-like Growth Factor 1 (IGF-1) that inhibits AFP gene expression.

  • Long summer day lengths stimulate growth hormone (GH) release from the pituitary, affecting AFP production.

  • Short winter days suppress GH release leading to reduced IGF-1 and inhibition of AFP transcription.

Terrestrial Environments and Freezing Strategies

• Terrestrial habitats face severe temperature fluctuations ranging from -20°C to -60°C.

• Need for cold acclimatization and other adaptations for overwinter survival includes:

  1. Supercooling methods (for freeze-susceptible species).

  2. Freeze-tolerance mechanisms.

Supercooling in Insects and Invertebrates

• Most terrestrial invertebrates, especially insects, adapt by supercooling.

• Supercooling Point (SCP): A threshold temperature at which spontaneous freezing (nucleation) occurs, e.g., summer SCP -6°C, winter SCP -25°C.

Supercooling Mechanisms

• Methods to enhance supercooling abilities include:

  1. Production of polyols (e.g., glycerol, sorbitol).

  2. Utilization of antifreeze proteins.

  3. Removal of ice-nucleating agents (INAs).

Polyols in Cryoprotection

• Small molecular weight antifreeze agents like polyols function to lower freezing points and SCPs through colligative methods, although they can increase overall osmolarity and be toxic.

Antifreeze Proteins in Terrestrial Invertebrates

• AFPs found in lower invertebrates (insects, spiders) operate similarly to those in polar fish, demonstrating notable thermal hysteresis activities without raising osmolarity.

• Sustained production of AFPs may occur in response to photoperiod changes.

Ice Nucleating Proteins (INPs)

• Specific proteins that promote ice formation at relative high, sub-zero temperatures enabling more controlled freezing processes, thus reducing osmotic shock risks.

• Applications in genetic engineering have been explored, focusing on recombinant bacteria containing INP genes.

Recrystallization Processes

• Critical during thawing, as larger ice crystals can damage cells via membrane shearing.

• AFPs can impede recrystallization, mitigating detrimental impacts and presenting commercial interest for application in preservation technologies.

Strategy Switching between Freeze Tolerance & Susceptibility

• Certain species exhibit capabilities to alternate between states of freeze tolerance and susceptibility based on environmental factors.

• Common attributes among these species include glycerol and AFPs, yet variations in ice nucleating proteins exist.

Research Insights on Insect Antifreeze Proteins

• Investigated the inhibition of ice recrystallization and the role of AFPs in protecting against ice growth and damage.

• A novel Type 3 AFP was isolated from Tenebrio and further studies illuminated its multigene family evolution.

Evolution of Antifreeze Proteins in Notothenioids

• Figures (9.30, 9.31) illustrate evolutionary pathways and structural gene correspondences of AFGPs in Antarctic and Arctic fish species.