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Adaptation of Poikilotherms to Subzero Temperatures

• Overview of Adaptation Needs

  • Importance of adaptive mechanisms previously discussed

  • Necessity of preventing injury from ice formation during freezing

Freeze Damage Mechanisms

• Causes of Freeze Damage

  • Primarily from cell dehydration

  • Most animals cannot withstand ice formation within cells

  • Intracellular ice crystals are lethal

  • Extracellular freezing occurs, but it leads to:

  • Damage due to dehydration of cell water

  • Formation of ice in extracellular fluids

Mechanisms of Cell Dehydration

• Processes Leading to Cell Dehydration

  • Lowered water content within the cell reduces volume

  • Increased concentration of solutes leads to:

  • Precipitation of solutes

  • Rupturing of cellular membranes

  • Denaturation of proteins

  • Interactions among proteins increase

Mechanisms to Prevent Injury from Subzero Temperatures

• Adaptation Mechanisms

  1. Behavioral Avoidance

  2. Rapid Cold Hardening: avoidance of cold shock

  3. Cold Acclimatization

  • A) Avoid ice by increasing supercooling ability (for freeze-susceptible species)

  • B) Species become freeze-tolerant

  1. Developmental Preparedness

Freeze Challenges Among Animals

• Factors Influencing Freezing Issues

  • Animals' blood osmolarity in relation to their environment

  • Isoosmotic organisms to seawater are unlikely to freeze unless the entire sea freezes

  • Hyperosmotic organisms to freshwater tend not to freeze

  • Saltwater teleosts are hypo-osmotic to seawater, which raises their freezing point above seawater

  • Terrestrial creatures facing subzero temperatures also risk freezing

Marine Teleost Adaptation Strategies

• A) Supercooling to -1.9°C

  • Safe unless they encounter ice

  • Behavioral avoidance through migration to deeper waters

  • Fish near ice are likely to freeze

• B) Freezing Point Depression

  • Some teleosts depress their freezing point below seawater's, but this process is complex:

    • Encounter issues with enzymes and renal functions when trying to become isoosmotic with seawater

    • Teleosts aim to remain hypo-osmotic and avoid increasing glucose or glycerol concentration

    • Utilize Antifreeze Proteins (AFPs) to achieve freezing point depression

Antifreeze Proteins (AFPs)

• Discovery and Characteristics

  • First identified: Antifreeze Glycoproteins (AFGPs)

  • Structure:

  • Comprised of repeating tri-peptide chains with carbohydrate groups attached

  • At least 8 variants of AFGPs exist in different species, varying in the number of repeating units

  • Some substitutions in AFGPs include proline for alanine in types 7 and 8

• Mode of Action

  • AFPs do not lower freezing points through colligative means, differentiating them from lower molecular weight antifreezes

  • They inhibit ice growth by directly binding to existing ice structures (adsorption to ice)

Thermal Hysteresis Produced by Antifreeze Proteins

• Definition

  • Thermal hysteresis refers to the difference between melting point and freezing point in solutions

  • Example:

  • For a crystal, melting point ($M<em>p = -0.9°C$) and freezing point ($F</em>p = -2.5°C$)

  • Thermal hysteresis ($TH = 1.4°C$)

• Types of Antifreeze Proteins

  • AFPs are classified as Thermal Hysteresis Proteins (THPs)

  • THPs most likely coat ice crystals

  • Possess an Ice Binding Domain (IBM) for effective adsorption

Diversity of Antifreeze Proteins

• Types of Fish AFPs

  • AFGPs (common in Antarctic cods)

  • Type 1 AFP (found in winter flounder): Alanine-rich and alpha-helical

  • Type 2 AFPs (from sea ravens): Cysteine-rich

  • Type 3 AFPs (from eel pouts): Non-alanine, non-cysteine rich

  • Type 4 AFPs (from long-horned sculpin): Glycine-rich

• Genetic Insights

  • Many of these AFP genes have been cloned revealing multigene families

  • Current structure-function studies focus on which features and domains enable ice binding and growth inhibition

Seasonal Changes in Antifreeze Protein Production

• Data Representation

  • Seasonal variations in antifreeze protein (AFP) production in winter flounder

  • Use of graphical data to depict changes in AFP concentration and freezing point over the seasonal calendar

Seasonal Regulation of Antifreeze Proteins

• Regulatory Mechanism

  • Seasonal changes in the expression of antifreeze proteins regulated by IGF1

  • Long day lengths trigger growth hormone (GH) release from the pituitary gland

  • GH stimulates the liver to produce IGF1, which inhibits factors that enhance AFP gene transcription

  • Short day lengths reduce GH secretion and hence IGF1, leading to reduced AFP production

  • Diurnal effects on AFP expression places importance on photoperiod

Classification of Antifreeze Proteins

• Summary of AFP Classifications

  • Categorizes fish and insect antifreeze proteins and peptides by type and structure

  • Different types characterized by variable sequences and structures, indicative of evolutionary adaptations

Terrestrial Environment Adaptations

• Challenges of Extreme Temperatures

  • Characteristic low temperatures ranging from -20 to -60°C

  • Necessity for both cold acclimatization and overwintering adaptations

  • Two fundamental strategies:

  1. Supercooling for freeze-susceptible species

  2. Freeze Tolerance

Supercooling Adaptations

• Characteristics of Supercooling

  • Supercooling point (SCP) relates to spontaneous nucleation temperatures

  • Variation from 0°C to -40°C

  • Summer SCP around -6°C; Winter SCP may drop to -25°C

• Cooling Curve Description

  • Illustrates the relationship between body fluid cooling and supercooling, as crystallization leads to heat release

Methods to Enhance Supercooling Ability

• Strategies for Increasing Supercooling

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

  2. Antifreeze Proteins (AFPs)

  3. Removal of Ice Nucleating Agents (INAs)

Polyols and Their Effects

• Role of Polyols

  • Small molecular weight antifreezes (e.g., glycerol, sorbitol) reduce freezing points on a colligative basis

  • Also effective in reducing SCPs

• Regulation Mechanism

  • Often influenced by temperature via changing biosynthetic pathways

Antifreeze Proteins (AFPs) in Terrestrial Organisms

• Functionality Similar to Polar Fish

  • AFPs cause thermal hysteresis activity, depress freezing and supercooling points without increasing osmotic pressure

  • Found extensively in lower invertebrates and plants

• Insect AFP Potency

  • Insect AFPs often exhibit greater potency for thermal hysteresis than fish AFPs

Removing Ice Nucleating Agents (INAs)

• Process of Removing INAs

  • Critical for achieving supercooling; involves:

  • Clearing the gut pre-winter

  • Eliminating biological nucleators

  • Masking INAs to facilitate supercooling

Successful Supercooling Strategies

• Multi-Faceted Approach

  • Integration of polyols, AFPs, and INAs removal or masking

  • Essential for freeze-susceptible species to remain supercooled without transitioning into a frozen state

Freeze-Hardy Species and Their Strategies

• Freeze Tolerance Defined

  • Some species (e.g., certain frogs and turtles) can tolerate freezing despite dehydration

  • Use of cryoprotectants to fend off desiccation

• Concept of SCP and LLT

  • In freeze-tolerant species, the SCP is higher than the lower lethal temperature (LLT)

Mechanisms of Freeze Tolerance

• Cryoprotectant Production

  • Utilization of small molecular weight cryoprotectants like glycerol to stabilize cells by:

  • Reducing osmotic gradients

  • Maintaining cell volume

• Characteristics of Cryoprotectants

  • Glycerol penetrates cells and increases solution viscosity, minimizing ice formation and osmotic stress

Ice Nucleating Proteins

• Function

  • Control the freezing process by promoting extracellular ice formation at higher subzero temperatures to prevent osmotic shock

  • Identifying and cloning genes from ice-nucleating bacteria assist in developing biological solutions (ice-minus bacteria)

AFP Function in Cryoprotection

• Recrystallization Protection

  • During thawing, larger ice crystals may grow at the expense of smaller ones, causing damage to membranes

  • AFPs block excessive recrystallization, offering a level of cryoprotective support

• Commercial Applications

  • Techniques derived from understanding AFP mechanisms may have practical utility

Switching Between Freeze Tolerance and Susceptibility

• Species Flexibility

  • Evidence indicates some species can shift between states of freeze tolerance and susceptibility depending on environmental conditions

• Common Mechanisms in Switching

  • Presence of cryoprotectants and AFPs, enabling regulatory mechanisms due to environmental factors such as photoperiod or hormonal responses

Variations in Overwintering Mechanisms

• Research Insights

  • Induces variation in mechanisms with documentation on ice nucleation influence and the biological reasons for shifts in strategies

• Understanding Switch Mechanisms

  • Ongoing studies investigate the unpredictability and adaptability of overwintering mechanisms among species.

Research on Insect Antifreeze Proteins

• Personal Research Contributions

  • Investigating the inhibition of ice recrystallization influenced by AFPs and polyols

  • Isolation of a novel Type 3 AFP from Tenebrio, highlighting its gene relations within a multigene family

  • Focus on structure/function studies related to AFPs and their thermal hysteresis capabilities.