Zoology Week 3 - Comparative Ecophysiology - 7 October 2024 PDF

Zoology Week 3 ============== Comparative Ecophysiology ========================= 7^th^ October 2024 ================== **Artic Tundra Zone** - Animals (48 spp.) Terrestrial e.g., artic fox, hare, ptarmigan, reindeer - Numerous migratory birds - Decomposed biomass stored as CH4 & CO2...

Zoology Week 3 ============== Comparative Ecophysiology ========================= 7^th^ October 2024 ================== **Artic Tundra Zone** - Animals (48 spp.) Terrestrial e.g., artic fox, hare, ptarmigan, reindeer - Numerous migratory birds - Decomposed biomass stored as CH4 & CO2 = carbon sink - The ecotone (or ecological boundary region) between the tundra and the forest is known as the tree line. **Taiga (Boreal Forest in N. America)** - Flora consists mostly of cold-tolerant evergreen conifers with needle-like leaves, such as pine, fir, and spruce - Growing season 130 days - Fauna includes woodpeckers, hawks, moose, bear, weasel, lynx, fox, wolf, deer, hares, chipmunks, shrews and bats. **Two main strategies for cold tolerance in terrestrial polar invertebrates** - Freeze-tolerant: - Animals endure ice formation in extracellular body fluid compartments - Freeze-avoiding: - Species possess physiological mechanisms that promote extensive supercooling of body fluids throughout the winter **Collembola** - Species living predominantly on the ground surface or in vegetation (epigeic species) - Relatively impermeable cuticula and are typical freeze-avoiders with high capacity for supercooling - Species inhabiting deeper layers of the soil - Little cuticular resistance to desiccation -- alternative strategy termed cryoprotective dehydration **Cryoprotective Dehydration** - Involves water vapour pressure (WVP) between ice in the soil and the supercooled haemolymph which drives a net outflux of water vapour - The difference in WVP is so large it results in water loss of organism - Continuing until pressure of body fluids equals that of the surrounding ice - This response eliminates internal body ice formation **Thermal hysteresis (TH):** - The separation of the melting and freezing temperature is usually referred to as thermal hysteresis, and the temperature of ice growth is referred to as the hysteresis freezing point. - The hysteresis is supposed to be the result of an adsorption of antifreeze proteins to the crystal surface. - Analogy: rock salt on roads to lower freezing point **Ice Binding Proteins** - IBPs adsorb to the surfaces of ice crystals and prevent water molecules from joining the ice lattice at the IBP adsorption location. - Ice that grows on the crystal surface between the adsorbed IBPs develops a high curvature that lowers the temperature at which the ice crystals grow, a phenomenon referred to as the Gibbs-Thomson effect. - This depression creates a gap (thermal hysteresis, TH) between the melting point and the nonequilibrium freezing point, within which ice growth is arrested. MaAFP has the attributes of a hyperactive antifreeze protein in having high thermal hysteresis activity at low protein concentrations **Temporal metabolic rate of Antarctic Gomphiocephalus hodgsoni (Collembola)** - Substantial spatial and temporal environmental variation - Microhabitat temperature fluctuations (\>30°C) - Limited resource availability for extended periods - Give rise to slow growth and protracted live cycles - Needs to respond to prime periods - Daily (short term) -- *Cryptopygus antarcticus* supercooling in hours - Seasonal (intermediate) - Evolutionary (long term) - *Cryptopygus antarcticus* Metabolic cold adaptation (MCA) **Antarctica's only endemic insect -- Antarctic midge, Belgica antarctica** - Terrestrials maritime Antarctica is among the most extreme yet simplified ecosystems - Climate change and anthropogenic impacts, influence of abiotic and biotic factors on species distribution - Intense abiotic stressors - E.g., low temperature, limited water availability and variable nutrient levels, UV radiation **Antarctic midge, (Diptera: Chironomidae)** - Adults are flightless, endemic, live 10-14 days - Adult emergence is a synchronous event occurring in early summer & mate in swarms - Smallest insect genome - Larvae of midges are non-selective feeders on decaying plant matter and terrestrial algae - 4 instars and can overwinter in any instar -- 2 years development - Egg covered in anti-freeze / anti desiccation jelly - Larvae are Freeze-tolerant and endure ice formation in extracellular body fluid compartments **Multi-level analysis of reproduction in an Antarctic midge identifies female and male accessory gland products that are altered by larval stress and impact progeny viability.** - Eggs are encased in a hygroscopic gel -- constituents unknown - Secreted by the female accessory gland - In other dipterans e.g., sperm viability, antimi - Materials transferred from the male *B. antarctica* accessory gland are unknown - Other dipterans males produce spermatophores - Male, accessory gland-specific secretions include steroid hormone, 20-hydroxy-ecdysone, a mating plug, promote copulation, enhance egg production, and even trigger egg laying **Gene enrichment of females, males & larvae** - Gene ontogeny (GO): - Molecular Function: - Male, accessory gland-specific secretions include steroid hormone, 20-hydroxy-ecdysone, a mating plug, promote copulation, enhance egg production, and even trigger egg laying - Cellular Component: - Male, accessory gland-specific secretions include steroid hormone, 20-hydroxy-ecdysone, a mating plug, promote copulation, enhance egg production, and even trigger egg laying - Biological Process - The larger processes by multiple molecular activities. Examples of broad biological process terms are DNA repair or signal transduction. **Genome: Tissue, larvae & adults, specific genes that have significantly higher expression** - 392 male-enriched genes (few for accessory glands) - 1825 female-enriched genes (few for accessory glands) - 862 genes enriched in larvae - Gene Ontology categories enriched in each stage included: - Carboxylic acid biosynthesis in males - DNA repair in females - Aminoglycan metabolism in larvae **Impact of larval dehydration stress on female fertility?** - Re-analysed results of RNA-seq study on dehydration and cryoprotective dehydration of B. antarctica - Increased expression of genes in autophagic pathways - Decreased levels of those linked to carbohydrate energy metabolism - What about impact on the female accessory gland? - Gland protein= larval serum protein significantly reduced expression - Protein in gel. Serves as protein reserve for subsequent dev stages - What does dehydration-evoked suppression in transcripts cause? - Reduced egg out- put - Reduced amounts of materials invested in the progeny at birth Ecophysiology of Polar Bears (Ursus maritimus) ---------------------------------------------- - Genetic research confirms polar bears evolved from brown bears approximately 500,000 years ago - Polar bears are K-selected (low reproductive rate) - Females mature and breed at 4 to 5 years - Litter size one to three cubs, two bubs being most common - Cubs typically remain with their mother until 2 years old - Minimum reproductive interval for adult females with a successful litter is 3 years - Females enter dens between September and December - A successful denning period requires adequate: - Thermal protection - Time for cub maturation - Lack of natural or human disturbance - Security from predation - There was a significant regional difference in polar bear diet composition between the three subpopulations - Polar bears in Gulf of Boothia consumed a higher proportion of ringed seal compared to Baffin Bay and Lancaster Sound - Bearded seal in higher proportions in Gulf of Boothia and Lancaster Sound - Harp seal consumption was highest & most frequent in Baffin Bay - Beluga whales had the third highest dietary contribution in all three subpopulations - Sex and age class no effect on polar bear diet composition in Gulf of Boothia or Lancaster Sound - Baffin Bay, bearded seal consumption was significantly higher in adult females than in adult males and younger age classes - But caused by 3 out 10 individuals **Thyroid hormones (TH)** - Thyroid hormones (THs) are essential for artic animals - THs have ubiquitous roles, controlling thermoregulation, metabolism and reproduction - Study aimed at examining the seasonal variations in plasma THs in relation to body condition, fasting state (using plasma urea to creatinine ratio (UCR). - Creatinine is a breakdown product of creatine phosphate from muscle and protein metabolism **Results: Body mass & season** - Restricting GLMM modelling analysis to females caught both during spring and autumn of the same year (N = 32 occurrences including one female caught 4 times) - A highly significant effect of season (i.e., time) was found - females being on average 29 kg heavier in September (arctic winter) compared to April (arctic spring) **Results: Body condition index** - BCI, which is a more accurate indicator of condition than body mass and influenced by location, season & status - Females in the North West of Svalbard showed poorer body condition (lower BCI values) than females sampled in other areas - BCI was lower in females with COYs compared to solitary females (no difference between YRLS & solitary) **Thyroid hormones (TH) Method** - Concentrations of THs in plasma - total tri-iodothyronine TT3; free tri-iodothyronine FT3; total thyroxine TT4; free thyroxine FT4 - were simultaneously measured - Radioimmune assay solid-phase ^125^I (RIA) kits (Coated tube) Quantified using a Cobra Auto-Gamma counter - Calibration curves from analysed calibrators were generated from the gamma counter software **How do radioimmune assays work** - Rаdiоimmunоаssаy technique (RIА) is a very sensitive in-vitrо technique used to measure the соnсentrаtiоn of antigens (e.g., hormone levels in the blооd) using antibodies directed аgаinst these antigens. - RIA is performed by using antibody-antigen binding and radioactive antigen - The basic principle is a competitive binding reaction, where the analyte (antigen e.g., thyroid hormone) competes with radio-labelled antigen for binding to the fixed amount of antibody or the binding sites of the receptor. **Polar bears, disease and climate change** - As air and water temperatures increase, species previously non-resident in Arctic waters are extending their ranges northward, leading to greater species overlaps and a concomitant increased risk of disease transmission - Potential for mortality events. - Of concern are *Brucella, morbillivirus, influenza A* **Polar bear pathogen seroprevalence** - Causes include: - Warming temperatures - Changing precipitation regimes - Arctic warming twice as fast as the rest of the planet - Potential to increase: - Pathogen persistence - Prevalence - Emergence - Transmission **Francisella tularensis** - Transmission occurs through several routes, including direct contact with infected host fluids, vector bites and through ingestion of contaminated food and water - A zoonotic gram-negative intracellular bacterium largely distributed in the Northern hemisphere - In Europe natural foci of tularaemia are situated in three large ecological areas: - Boreal forest taiga - Temperate broadleaf and mixed forest - Temperate grassland and shrubland - *Francisella* *tularensis* can survive weeks in cold, moist environments including water, soil, hay, straw and decaying animal carcasses. **Immune system cell biology** - The blood plasma aka serum is an amber-colored, protein-rich liquid - Immunoglobulins play a key role in the body's immune system. - They are antibody proteins produced by specific immune cells - Immunoglobulins, including IgM antibodies, are found in all jawed vertebrates (gnathostomes) that diverged in evolution from jawless fish (agnathans) approximately 550 million years ago **IgM (macro-immunoglobulin) antibodies** - IgM -- 1^st^ response immunoglobulin on exposure to foreign bodies - Providing short-term protection whilst the body makes other antibodies - In mammals, B cell (lymphocyte) development occurs in an ordered manner: - Foetal liver during embryonic development -- detected at 20 weeks in humans - Bone marrow - Peripheral lymphoid tissue in adults - Produced by plasmas blasts residing in the spleen **IgG (immunoglobulin G) antibodies** - IgG is a monomer with two antigen binding sites - IgG formed by plasma B cells fighting infections from bacteria and viruses. Most of the immunoglobulins in your blood are IgG - Your body keeps a "blueprint" of all the IgG antibodies you have made - If exposed to the same germs again, your immune system can quickly make more antibodies - IgG is the only immunoglobulin that crosses the placenta (humans) **Male vs Female & pathogens** - Males were 1.82 x more likely to be seropositive for *Trichinella* spp. than females, - Females were 4.95 x more likely to be seropositive for *B. bronchiseptica* - Both these pathogens prevalence increased with bear age - Conflict bears (previously captured in human settlements) - Increased 2.24 x for *Trichinella* & 1.97 x less likely for *F. tularensis* **Climate & pathogen prevalence** - Summer Temp: - Warmer summer temperatures was a top factor in the seroprevalence of Trichinella spp. And B. bronchiseptica - Winter Temp: - Warmer winters - Top factor for Trichinella - Precipitation: - Wetter summers within dryer years increased the likelihood of polar bears seropositive for T. gondii. **Additional explanation on RIA** - A known quantity of an antigen is made radioactive gamma-radioactive isotopes of iodine attached to tyrosine. - This radiolabeled antigen is then mixed with a known amount of antibody for that antigen, and as a result, the two chemically bind to one another. - Then, a sample containing an unknown quantity of that same antigen is added. - This causes the unlabeled (or \"cold\") antigen from the serum to compete with the radiolabeled antigen (\"hot\") for antibody binding sites. - As the concentration of \"cold\" antigen is increased, more of it binds to the antibody, displacing the radiolabeled variant, and reducing the ratio of antibody-bound radiolabeled antigen to free radiolabeled antigen. - The bound antigens are then separated from the unbound ones, and the radioactivity of Ecophysiology of Hibernating Bears ---------------------------------- - Brown bear (Ursus arctos arctos) - Largest terrestrial carnivores in Central Europe - Height between 1.70-2.20 m - Weight varies between 100-350 kg - 75% nutritional needs from plant matter - 20,000 calories a day for sufficient fat for winter - Breeding May to July (polygamous) - Fertilized egg floats freely in the female's uterus for up to five months, implanting itself only at the beginning of hibernation - January and March, two to three cubs are born in the winter den - Hibernate November to March, sleep continually and do not feed or drink water, urinate or defaecate - Scandinavian brown bear (Ursus arctos arctos) - During hibernation metabolism is depressed - Body temperature decreases to varying extents - And a suite of cellular and physiological changes culminates in the ability to survive periods of food scarcity - Medically, humans who have sedentary lifestyles are prone to: - Heart disease - Type 2 diabetes - Kidney disease - Metabolome: - Total number of metabolites present within an organism, cell or tissue - Metabolite: - Substance formed in or necessary for metabolism - Metabolomics: - Emerging field that quantifies numerous metabolites systematically - Proteome: - Number of proteins present in an organism, cell or tissue - Proteomics: - Study of the structure and function of proteins, including the way they work and interact with each other inside cells Differences in plasma proteins (proteome) - Protein concentration was significantly higher in winter - Innate antimicrobial proteins protecting health were significantly increased in hibernation - Down-regulating complex multiprotein cascades and up-regulating a small number of generalist and principal effector proteins - Very few intracellular proteins released from bone and muscle tissues Examples of 154 proteins identified - SHBG - sex hormone-binding globulin isoform 1: - Functions as an androgen transport protein but may also be involved in receptor mediated processes. - Each dimer binds one molecule of steroid. - Specific for 5-alpha-dihydrotestosterone, testosterone, and 17-beta-estradiol. - Regulates the plasma metabolic clearance rate of steroid hormones by controlling their plasma concentration. - CAMP - cathelicidin antimicrobial peptide: - Binds to bacterial lipopolysaccharides (LPS), has antibacterial activity. - CA2 --carbonic anhydrase 2: - Essential for bone resorption and osteoclast differentiation - ALB -- serum albumin: - Good binding capacity for water, Ca2+, Na+, K+, fatty acids, hormones, bilirubin and drugs - Main function is the regulation of the colloidal osmotic pressure of blood - Major zinc transporter in plasma, typically binds about 80% of all plasma zinc. Targeted mass spectrometry identification and quantification of metabolome - Targeted metabolomes of phospholipids, hexose sugars, and amine-containing compounds - Carnitines e.g., - ammonium compound (amine compound) - play a critical role in energy production. - It is an essential cofactor that helps transport long-chain fatty acids into the mitochondria so that they can be oxidized to produce energy in the form of adenosine triphosphate (ATP) - Phosphatidylcholines e.g., - major components of biological membranes and pulmonary surfactant - essential component of the very low-density lipoprotein (VLDL) complex that facilitates the transport of triacylglycerols out of the liver - Sphingomyelins e.g., - sphingolipid - involvement in the regulation of endocytosis - receptor-mediated ligand uptake - ion channel and G-protein coupled receptor function - in protein sorting - Component of myelin sheath & cell membranes Summary proteome and metabolome - Albumin which constitutes 50% of plasma protein, was increased to 1.18-fold in hibernating bears - Higher viscosity of plasma obtained from hibernating brown bears - levels of four protective anti- microbial proteins increased - Haptoglobin-2 (4.7-fold) - antimicrobial, antioxidant, and a recycler of iron (bind potentially harmful HB released naturally from RBC & protects kidney) - Lysozymes (2.7-fold ) - primarily a bacteriolytic function; those in tissues and body fluids are associated with the monocyte-macrophage system and enhance the activity of immuno-agents. - N-acetylmuramoyl-L-alanine amidase (2.2-fold) -- Possibly digests biologically active peptidoglycan into biologically inactive fragments. Defense response to Gram-positive bacterium but not a bacteriolytic. - IGA1 - is the major immunoglobulin class in body secretions. It may serve both to defend against local infection and to prevent access of foreign antigens to the general immunologic system 1. Hibernating bears do no defecate or urinate 2. Therefore, amino acids need recycling for protein synthesis during hibernation 3. This means a requirement for nitrogen to be retained in a non-toxic form (ammonia). 4. Amino acids and biogenic amines slowed increased levels of molecules containing two or more nitrogen atoms 5. Intracellular proteins generally have short half-lives 6. However, that can be increased in hibernation by 7. Increased Alpha-2-macroglobulin an extracellular mainly known for its role as a broad-spectrum protease inhibitor 8. Protein degradation lowers in reduced temperatures 9. Cardiac output reduced by 25% - Hibernating bears are dehydrated as shown by increased haematocrit - ratio of the volume of red blood cells to the total volume of blood. - Hibernating bears exclusively get water from fat metabolism - Water loss in hibernating bears comes from pulmonary ventilation - Importantly energy conservation is is vital in hibernation. - Under non-stressed conditions protein synthesis consumes more than half of organismal energy. - The rate of hibernating bears protein synthesis is low as shown in previous slides - Protein synthesis \>90% of cells energy whereas increased levels of mRNA can respond quickly and is energy efficient Steroid Hormones - Scandinavian brown bears hibernate October/November to April - Proposed that mediators of hibernation are probably endocrine regulators - Sex steroid sex hormone -- binding globulin (SHBG) is the protein that increases the most in blood plasma between active and hibernation states - There are five major classes of steroid hormones: - Progestogens - Glucocorticoids - Mineralocorticoids - Corticosteroids - Androgens - Estrogens Steroids are lipophilic and bind to carrier proteins - Corticosterone-binding globulin (CBG) -\> Binds to glucocorticoids and progesterone - Sex steroid sex hormone binding globulin (SHBG) -\> Binds to active sex steroids - Albumin -\> Binds to all classes - Carrier proteins act as regulators as they are lipophilic - Cholesterol is the starting material for all the classes synthesis - So, it is interesting to see steroid hormone levels and the levels of two of their binding proteins, both in the active and hibernation states Adrenal gland: Example hormone pre-cursors also measured - 17-hydroxyprogesterone -- converts to cortisol - Cortisol regulates metabolism and the immune system - Stress hormone: fight or flight --glucocorticoid class - 11- deoxycorticosterone -- precursor to aldosterone - Mineralocorticoid -- adrenal gland - Salt retaining - Androstenedione - Pre-cursor in synthesis of testosterone, androgens & estrogens like weak estrone What does this mean - Cholesterol - Several important functions including a precursor for steroid hormones - Cholesterols main storage area is adipose tissue - This may be the reason for increased concentrations due to hibernation lipolysis - In addition, cholesterol & triglycerides are transported by lipoproteins - Due to much lower metabolic rate in hibernation, suggested that a lower rate of lipoprotein catabolism Progesterone - Increased in hibernation in both male and female bears - Postulated increase due to being a precursor for other steroid hormones - Examples: - Glucocorticoids (increased in hibernation) - Aldosterone (mineralocorticoid) Sex hormone -- binding globulin (SHBG) - Binds tightly to the steroid's testosterone, oestradiol and DHT and transports them in the blood in an **inactive form** - In humans, high SHBG lowers risk of various diseases, such as metabolic syndrome, diabetes, cardiovascular diseases and muscle weakness  - Sex hormone activity appears to be a prerequisite for initiation of hibernation in e.g., hamsters, hedgehogs, and ground squirrels. - Is function to quench sex steroid activity in bears during hibernation? What is echocardiography? - Properties of sound waves - Speed of acoustic wave depends on density & resistance of medium - Velocity = wavelength (mm) x frequency (Hz - Number of waves that pass a given point in one second) - Dense media transmit mechanical wave with greater speed - Shorter wavelength means reduced tissue penetration Velocity of blood flow - Doppler effect or shift -- high frequencies towards and low frequencies when moving away. Change in wavelength caused by motion of source - In tissue mean sound velocity 1540 m/s - Ultrasound of red blood cells change between transmitted & received frequency on return