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Marine Molecular - Coggle Diagram

- - - - Measures population differentiation between sample sites due to genetic structure (genetic diversity), FST is usually calculated pairwise where every sampling site is compared to all other sampling sites (if there was low differentiation the value would be 0) it is usually set in a table
    - - A method of estimating population differentiation from molecular data, uses phi-statistics which summarise the degree of differentiation between population divisions (haplotype frequency and divergence), allows hypothesises to be tested
    - - A diagram of haplotype relationships including possible mutation pathways in between them, there is a map with colour-coded spots on it that matches the network, this shows sampling sites, each circle is one haplotype, size of the circle is linked to frequency, connecting lines show mutational pathways (lengths of the lines mean genetic distance) and black squares or any form on interruptions on the line means missing data
    - - Uses Da distance (the number of net nucleotide substitutions per site between populations so intra-population variability is taken into account
- - - - Cryptic/ sibling species are pairs or groups of species that are sympatric (live in the same geographical habitat), closely related and morphologically identical but are reproductively isolated, these species can be revealed by using markers like mtDNA, microsats or nuclear DNA, and can be used to help manage fisheries so they don't accidently fish a species to extinction
      - Marine hybrids are a result from interbreeding of two separate species resulting in the possibility of one species forming
      - Sub-species are species that are partway through speciation (the process is slow) They have a unique geographic range or habitat and are genetically distinguishable, they don't exhibit reproductive isolation and can produce fertile offspring however there may be some fertility or survival issues in some of the offspring.
      - Non-invasive sampling: Environmental DNA (eDNA) - Genetic material obtained directly from environmental samples(seawater etc) without any obvious signs of biological material
        
        Pros: No need for visual ID, identify cryptic species at juvenile stages, alternative to taxonomic experts, cost efficient, sensitive, always improving, non invasive, easy to standardise sampling, can be used on extinct species
        
        Cons: Contamination, inhibition, not specific or too specific, high error rates, dilution in seawater, maintaining reliable results over temporal and spatial scales
      - Molecular identification techniques can be used for forensics for when protected species are fished and used in the food industry or used in the aquarium trade when they shouldn't be
      - Gut content analysis is when the gut is manually examined (this could be lethal and is definitely invasive) to see its contents and see what the species eat/ prefer to eat, one issue is digestive processes can interfere with this. Usually this process uses mtDNA as a marker
  - - - There are situations where this definition doesn't apply for example asexual organisms, hybrids, fossil application and there are some cases where isolated species have bred successfully
- - - - Transcription: Genes are transcribed in a 3 step process, initiation (RNA polymerase binds to the promotor region (where RNA polymerase starts transcription) and is promoted by transcription factors (proteins that bind to the promoter region and help initiate transcription, they recruit RNA polymerase)), elongation (adding complementary bases to the original DNA simultaneously producing 2 strands of pre-mRNA) and termination (RNA polymerase stops at the terminator).
        
        Splicing: pre-mRNA is produced from transcription and is then spliced (introns removed) then exons remain, introns are used for alternative splicing (introns can be used, cut or take exons with then to make different proteins (removing an exon makes a different protein), some introns are involved in regulatory processes that control transcription of DNA.
        
        Translation: Happens in ribosomes which have 2 subunits (18s (small subunit) and 28s (large subunit)), the subunits sandwich the mRNA and attract tRNA molecules that are attached to amino acids, the anticodon on tRNA is complementary to the codon on mRNA, the amino acids bond once next to each other and the the tRNA goes leaving an amino acid chain, the genetic code determines what amino acid is added.
        
        Post translation: Proteins can still be modified, enzyme reactions occur (enzymes are proteins), forming metabolites (the end products of intermediates of cellular reactions). Metabolome is the complete set of small-molecule chemicals found within a biological sample (this could be a cell, tissues, organs etc)
    - - Proteins are functional units, used to make structures, control chemical reactions and carry signals, they are large biomolecules, synthesized in ribosomes during the process of translation (triplets code for amino acids, amino acids make peptides which make proteins)
        
        Proteome - Full sector of proteins expressed buy the genome of a cell, tissue or organism at a specific time point
        
        Proteomics - allows high-throughput (processing rate) analysis for the study of protein mixtures in complex systems. Tools like ELISA, western blot, 2D gel electrophoresis (including DIGE) and mass spectrometry (overall these techniques are less developed than transcriptomics). Can be applied to recovery and analysis of proteins from the marine environment (eg looking at degradation of proteins) and verifying genomic findings (to see which genes were translated), western blots are used in medicine (eg diagnosing Huntingdons)
    - - DNA is a nucleic acid that has a code made of the nitrogenous bases adenine (A), guanine (G), cytosine (C) and thymine (T), it is a double helix that is made up of two strands, DNA (polymer) is made up of nucleotides (monomers) that have a phosphate group, a pentose sugar and a nitrogenous base, the nucleotides are bound by 5' 3' phosphodiester bonds which means they are linked by the phosphate group at carbon 5 of the pentose sugar and carbon 3 of another pentose (deoxyribose) sugar (the carbons are numbered from the oxygen clockwise), there will be a free 5' carbon at the top and a free 3' at the bottom, the strand attached has the backbone going the other way (3' at the top, 5' at the bottom). DNA replicates using semiconservative replication where one strand is old (used as a template) and one is new (made from free-floating nucleotides (nucleotides are complementary A forms 2 hydrogen bond with T and G forms 3 hydrogen bonds with C - this is how the 2 strands stay together)),
        
        A gene is a sequence of DNA that codes for a specific protein (in humans genes make up ~1.5% of the genome)
        
        Genome - All genetic information in an organism
        
        Overall the nucleotides make the DNA, the DNA make up the genes which are coiled around histones which form chromosomes which are in the nuclei of many cells and if you add up all the genes in that organism it is the genome
    - - RNA is single stranded, made of nucleotides like DNA however ribose sugars are used instead and instead of thymine there is uracil (U), it is produced from transcription of DNA and it is still complementary to DNA (adenine has hydrogen bonds with uracil)
        
        Transcriptome - Set of all RNA molecules produced in a cell or tissue at a given time (this means that it is constantly changing)
        
        Transcriptomics - methods of analysis of gene expression changes in a cell or tissue under different conditions. Can be applied to identifying mechanisms underpinning physiological functions, environmental monitoring and early warning system for chemicals, toxins and environmental change (temperature, ocean, acidification, hypoxia, nutrient depletion etc
      - Types:
        
        mRNA (messenger RNA) - carries a message, is produced in transcription and then undergoes translation
        
        tRNA (transfer RNA) - used in translation
        
        rRNA (ribosomal RNA) - used in structure of molecules and ribosomes
        
        ncRNA (non coding RNA)
        
        microRNA - regulatte transcription in genes
      - RNA isn't very stable, it forms a message that is required for expression in cells, it degrades rapidly by RNase enzymes so when used in the lab RNA samples need to be frozen (quick but thawing can cause problems) or be stabilised (which is safe but can interfere with some of the samples or not work)
    - - Quantitative real time PCR is used to quantify expression levels of a gene of interest in a given cell or tissue, knowledge of gene sequence is required beforehand, it also requires a specific sequence of primers and it combines amplification and detection (the amplification step happens in the presence of a DNA binding fluorescent dye)
      - PCR process: Heat DNA to 95C to unzip DNA, then reduce to 55C so primers stick to gene (annealing - primers bind to complementary single-stranded DNA templates, initiating DNA synthesis and ensuring specific amplification), then heated to 72C where polymerase adds nucleotides, 2 copies of genes are made an the process gets repeated. This is all done in a thermocycle which is a programmable heat block where samples are put in tubes and it takes it through the PCR temperature cycle
      - End point PCR is non quantitative (you can see the DNA is there but not how much of it) because it isn't exponential in the middle of the reaction (DNA increase levels vs cycles)
      - Eventually at the end of PCR reactions the number of DNA produced plateaus
    - - Metabolome - a set of naturally occurring small molecules (metabolites) that are present in blood, urine and tissue at any given time
        
        Metabolomics focuses on characterising and measuring the levels of thousands of metabolites like sugars etc
        
        Used in looking at toxicology, disease, animals response to external stimuli and identifying molecules that could be biomarkers to identify general ecosystem health
  - - - Targeted approach: qPCR (quantitative PCR) looks at quantifying gene expression. qPCR instruments measure product accumulation in every cycle by measuring fluorescence and it shows the cycle when fluorescence is first detected and when it crosses the threshold. Fluorescence increases as the dye was bound to DNA but is then released. In some cases housekeeping genes are used as control/ reference gene to compare your sample to.
        
        Primer, the sample, nucleotides (dNTPs), DNA polymerase and SYBR green (a fluorescent dye that fluoresces when bound to double stranded DNA) are added to a qPCR machine (it is like a thermocycler but it also detects fluorescence). So the sample undergoes regular PCR and once the DNA strands are made SYBR green binds and releases fluorescence which is then detected by the qPCR machine, from this a graph of fluorescence (y axis) and cycle (x axis) is produced where there's a threshold of fluorescence to show, the less amount of cycles for the threshold to be met the more DNA the sample has. A melt curve is produced on another graph to show if there are any contaminations, if there is one amplicon (DNA/ RNA getting amplified) there should be one peak on the curve if there are any more then there may be some contamination, there should also be no lines in the NTC (the area under the melt curve). Data is then analysed using the delta delta CT method to see if the results are significant.
        
        Before the actual process of qPCR can happen RNA needs to be isolated first and then checked for its purity (measured by nanodrop/spectrophotometer -it measures absorbance of UV light) and integrity (being whole, can be measured by bioanalyzer or gel electrophoresis (gel electrophoresis can also locate contaminants)
      - General approach
        
        RNAseq aka RNA sequencing (is NGS but focuses on RNA): The aim of this is to identify features of the transcriptome that are expressed differently between individuals or tissues. No prior knowledge of the genome is needed, it offers single nucleotide resolution making it possible to detect related genes, higher dynamic range of expression levels where transcripts can be detected, high reproducibility and lower technical variation. The same technological platforms are used as NGS.
        
        Step 2: Laboratory performance involves extracting RNA, library preparation involves RNA getting reverse transcribed into cDNA and then gets fragmented and amplified by PCR. The sequencing and imaging step then happens where there are fragments read by the sequence called reads, the reads overlap and assemble into contigs by looking for similarities, if the number of reads to make the sequence is higher it means it is expressed more.
        
        Step 1: First you need to design the experiment by selecting the library type (what you are going to sequence), the platform type based on the sample the sequencing depth (how many times you sequence the fragments) and the number of replicates (how many (if any) replicates are needed to find differences)
        
        Step 3: Data analysis involves quality control, read mapping, quantification (how many times each contig gets detected (counts)) and differential expression analysis (analysing read count data to discover quantitative changes in expression levels between experimental groups). From this visualisation tools like heat maps (a grid where the rows are area genes and the columns are samples and the colours of the shows an increase of gene expression (upregulation) or decrease of gene expression (down regulation)). There are also biological interpretation tools like Gene Ontology (GO) enrichment (looking at the database and comparing it to other genes to see if there's more expression) and KEGG (Kyoto Encyclopaedia for Genes and Genomes) pathways (a series of maps that represent the molecular interactions between different genes).
        
        Microarray
        
        Step 1: Microarrays (printer-like machine) consist of slides where bubbles of oligos (fragmented DNA) of a known sequence are printed
        
        Step 2: 2 samples are prepared by having their RNA extracted, and then the RNA gets reverse transcribed to DNA and then each sample has a different dye, the samples are then mixed (hybridised) and poured into the microarrays to bind to the oligo bubbles printed in step 1, if a lot of one colour is shown then it means that oligo has lots of expression in common with that sample as that sample is binding to it, sometimes there can be a mixture of the 2 samples and it shows as a mix of the 2
      - There are limitations to qPCR and microarrays that RNAseq overcome such as the requirement for previous knowledge of the genome sequence, risk of cross-hybridisation, limited detection and the fact it is difficult to compare expression levels across experiments
    - - Targeted approach: Western blots (used to look at how much protein is present in a tissue/ organism). Challenges: cost, lots of antibodies need to be produced and tested
        
        Step 1: Prepare the protein mixture and run it on a gel to separate the proteins (by electrophoresis so it is separated by weight)
        
        Step 2: Proteins that stick are transferred to a surface where antibodies that are complementary and detect the protein of interest are added. This should result in the antibody binding to the antigen on the protein. Then a secondary antibody coupled with a fluorescent or chemiluminescent signal that will be released once binding to the antibody that is bound to the target protein. While the signal does show it is a good opportunity to see where the protein is and how much of it there is.
      - General approach:
        
        2DE (2D gel electrophoresis) is gel electrophoresis ran twice in 2 dimensions (electrophoresis isn't used as much nowadays)
        
        Step 1: There is a strip of gel that has a pH gradient so the proteins separate themselves out to their isoelectric points (the pH where the protein has no electrical charge), proteins stop when they reach this point
        
        Step 2: The gel strip will then be attached on top of a larger gel section which then has a current ran through it resulting in proteins separating by size (small proteins move further) and you can still see what side is acidic and what side is basic. The proteins are then dyed with silver nitrate so scientists can see the spots/proteins
        
        DIGE is a variation of 2DE where 2 samples are dyed with different colours and were put in one gel however it wasn't as popular
        
        Mass Spectrometry
        
        Proteins are separated from organs, tissues or cells by one dimensional polyacrylamide gel electrophoresis or 2DE, then the proteins are digested into peptides, then chromatography happens in a column that has organic solvents and a gradient resulting in peptides getting eluted (extracted) out of the column, they are ionised by electrospray ionisation (high voltage applied to a liquid to make it an aerosol), ions are then transferred to a mass spectrometer (vacuum) and the mass to charge ratio is measured which can be compared against the mass spectra to work out the molecular weight of the peptides and then that can be used to identify the protein
    - - Targeted approach (involves a small number of genes/ is for a specific gene): Sanger sequencing: This focuses on genetics (studying specific or limited numbers of genes). Sanger sequencing uses the process of synthesis of DNA to find out the sequence of the fragment.
        
        Step 1: DNA polymerase and dNTPs (nucleotides) are added to a primer (a DNA fragment), the polymerase adds the dNTPs in a way that they are complementary to the sequence so that the DNA is copied. Fluorescently labelled ddNTPs (dNTPS but with no OH group on the third carbon meaning nothing can bind to it afterwards so it stops the rest of the backbone forming) are also added in with the dNTPS, so many different strand lengths are created and many copies of these are created, all having fluorescent bases
        
        Step 2: All of these fragments are ran in a capillary for electrophoresis (a current if ran through a gel and the DNA goes through the gel, the small DNA moves further and faster while the larger one moves slower and shows more resistance, the DNA moves because it has a slight negative charge. As the bases are dyed differently the sequence can be seen using technology and a graph-like image being produced showing what bases are expressed and where they are
      - General approach: Next generation sequencing (NGS): This focuses on genomics (studying the structure, function and diversity of genomes), this uses the massively parallel sequencing technique which is when separation, sequencing and detection steps are combined and many reactions happen quickly all at the same time so multiple fragments can be sequenced at once, so sample size increases, sequencing speed increases, it is more accurate and is cheaper
        
        Step 2: Library amplification: DNA is amplified to provide a cluster of identically sequenced molecules, this is done by PCR, this is done so the received signal from the sequencer is strong enough to be detected
        
        Step 3: Sequencing: Sequencing occurs via repeated cycles of nucleotide addition by an enzyme, detection of the incorporated fragments and washing steps, this can be done by different technological platforms. The sequence can then be detected by fluorescent labels attached to the nucleotide or the product of a nucleotide incorporation reaction involving a proton or pyrophosphate. After this a lot of data is produced which then needs to undergo bioinformatics analysis (using software tools to interpret biological data). As there is so much data many sequences of different parts are used they can be assembled together to get the consensus (the full sequence made from shorter sequences) sequence (consensus sequences are also known as contigs) made from sequences (short reads) without any repeats, this is done by looking for overlaps in the short reads (smaller sequences provided) and then putting them together. This process is called an assembly and it has 2 types de novo genome assembly (generating a new genome sequence from NGS data) and de novo transcriptome assembly (harder because data is more heterogenous (consists of different parts), there could be alternative splicing events and one output is a measure of transcriptome abundance). Another process related to assembly is alignment/mapping where you find the appropriate origin of a sequence in an existing transcriptome or genome. Another process linked to mapping is annotation which is marking specific features in a DNA, RNA or protein sequence with descriptive information about structure or function (this can be done using a database search).
        
        Step 1: Library preparation (a library is what you are going to sequence): Before library preparation starts the DNA or RNA are extracted from the cell and yield, cleanness, quality and purity are checked, only good DNA is used for library preparation. When library preparation happens RNA gets reverse transcribed into cDNA, regular DNA and amplicon DNA (source/product of replication events) as well as this mRNA get fragmented based on the generation and platform of technology used. Then DNA adapters are attached to the sequences to enable binding of library fragments to a surface (it can be microbead or glass)
    - - Targeted approach: Biochemical assays: Biochemical assays are laboratory methods that measure the presence or activity of specific biological molecules or reactions. They provide quantifiable data about substances like enzymes, proteins, and metabolites, which are essential components of all biological functions.
      - General approach: NMR spectroscopy and mass spectrometry: NMR (nuclear magnetic resonance) spectroscopy relies on detection and analysis of electromagnetic waves emitted by the nuclei of the sample from it getting disturbed, due to the unique spectra of the wave the sample can be easily identified
    - - Gene knockdown by RNAi (RNA interference) or dsRNA (double stranded RNA): RNA inhibits gene expression by degrading targeted mRNA molecules which results in a decrease in activity as they prevent translation which results in gene silencing. When double stranded RNA is exogenous (manipulated in a lab) the RNA can be imported into the cytoplasm and is cleaved (broken down) into siRNAs (small interfering RNAs) by an enzyme called dicer. siRNA is then integrated into in the RNA Induced Silencing Complex (RISC) which degrades the mRNA fragments and then from that you can look at the change in phenotypic change. This only decreases the mRNA it doesn't completely degrade it.
      - Gene knockout (and editing) by CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats): CRISPR is a naturally occurring genome editing system in bacteria and is used as an immune system, CRISPR complex is made up of sgRNA (single guide RNA) which recognises the target sequence (the one that needs to be edited or knocked out) and Cas9 endonuclease cuts the target sequence. Cas9 recognises a small sequence of DNA called PAM (Protospacer Adjacent Motif) which indicated the areas that need to be cleaved, this is done by Cas9 binding to the DNA which causes a break at the target site, the host cell will attempt to repair this but mutations are heavily likely to arise so transgenic (altered) DNA will be formed which will stop translation or result in a different protein being produced so a knockout happens regardless.
  - - - can refer to noxious (harmful) stimuli that an individual is exposed to, physiological and behavioural coping responses to stimuli, overstimulation of coping responses resulting in disease
      - Stress can be applied to environmental factors like temperature, hypoxia, pH, UV and pollutants
        
        When it comes to the effect of environmental factors, fitness is impacted as it can only be maximised in optimal conditions, there are windows of these factors where some can survive but only just and then there's smaller windows for thriving however responses to this depends on duration and magnitude. Some environmental factors can even effect species on a molecular basis for example temperature breaks bonds which denatures proteins, so the aim of a stress response would be to prevent cell damage by repairing proteins
        
        Stress response - a series of cellular and physiological mechanisms aimed at maintaining homeostasis
        
        Cellular stress response is triggered during severe acute stress which causes a proportional increase in macromolecular damage that disturbs cellular homeostasis, so the aim of cellular stress response is to restore structural and functional homeostasis in stressed cells and tissues, this involves repairing macromolecular damage to structures like DNA and proteins, growth arrest (activating cell cycle checkpoints) to save energy and prevent faulty DNA/ damaged genomes getting passed on, redirecting metabolic energy to macromolecular repair and finally programmed cell death (apoptosis). Stress levels are measured by looking at the cellular stress response
        
        Heat shock response is a quality control system that repairs and prevents protein unfolding
        
        Molecular chaperones - a large group of unrelated protein families whose role is to stabilise unfolded proteins, unfold them for translocation across membranes or for degradation and to assist in their correct folding and assembly
        
        Without the heat shock response the native proteins would have bonds break within them resulting in unfolding which means they lose their structure which results in them aggregating (clustering) with other unfolded proteins which causes toxicity
        
        The heat shock response is done by unfolded proteins undergoing Hsp (heat shock protein) mediated folding which can result in native protein formation or if that is unsuccessful the misfolded protein undergoes the ubiquitin proteosome system where a chain of ubiquitin proteins (ubiquitous proteins are designated and classified according to their molecular weight) bind to the misfolded protein (ubiquitination) labelling it for degradation by proteolysis at the proteosome, whatever smaller proteins are produced get recycled
        
        Heat shock proteins
        
        Hsps are molecular chaperones which assist in folding and transporting native proteins
        
        Expression patterns of stress induced molecular chaperones can be used too discern the vulnerability of tissues/ organisms to damage caused by stress
        
        Hsps are highly conserved across taxa, they are present in unstressed cells (these specifically are called constitutive (permanently expressed)), once they are expressed due to stress exposure they are known as inducible (aggregation is prevented)
        
        Hsp70 (70 because of its molecular weight) is a key protein used for assisting misfolded proteins or targeting malformed proteins for degradation
        
        Transcriptional activation of heat shock proteins
        
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        Thermal history can effect Hsp expression for example species in warmer environments produce Hsps at higher temperatures than species that live at cold environments, with species in different zones on the sea shore thermal tolerance varies so there is a wider window of tolerance for those that deal with extreme conditions
        
        Although HSP expression is plastic it does have its limits which results in species staying in boundaries of land they can handle. (chronic exposure can increase tolerance but it is limited)
        
        When the heat shock response does happen many proteins are downregulated aside from those that are involved in reparations
        
        Hypoxia
        
        Oxygen
        
        Oxygen is a diatomic molecule that makes up 21% of the atmosphere
        
        Oxygen is required by aerobic life (most life is aerobic), it is taken in through respiratory surfaces and transported (by blood) in respiratory pigments to cells or tissue for respiration to happen this process has a lot of reactions, oxygen is a terminal electron acceptor in the electron transport chain after respiration is complete ATP/energy is produced (32 in aerobic, 2 in anaerobic)
        
        There are over 400 oxygen dead zones around the world formed due to eutrophication and climate change (the temperature increase effects O2 solubility), these sites are increasing exponentially. There are oxygen minimum zones (OMZ). Without oxygen there is no aerobic life, no aerobic respiration so ATP falls (this means that less energy is spent on growing, feeding or fitness whatever is left is spent purely on surviving) energy decreases and without oxygen the cells reach disruption ion homeostasis which causes damage that makes cells and tissues die
        
        Hypoxia effects taxonomic groups differently the most sensitive are crustaceans, fish have sublethal responses at the highest O2 concentration, ontogenic shifts can happen (larvae are more sensitive), the difference of thresholds shows there must be some form or adaptation or mechanism that helps different species survive hypoxia
        
        ROS (reactive oxygen species) are toxic by-products of mitochondrial metabolism produced in the electron transport chain, it can be detoxified by antioxidants, increased ROS or unpaired antioxidants can lead to oxidative stress (imbalance of ROS and antioxidant) which can result in damaged DNA, lipid peroxidation (lipid degradation) and protein carbonylation (oxidation of the side chains of proteins). In small amounts ROS are good for signalling
        
        Types of antioxidants
        
        High molecular mass antioxidant enzymes (directly neutralise ROS) includes superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GP) and peroxiredoxin system
        
        Low molecular mass antioxidants (act as radical scavengers and interrupt radical damage) includes metabolites and vitamins
        
        ROS levels are altered during hypoxia as metabolic activity is reduced however ROS increases as mitochondrial complex III is altered it has been suggested that mitochondria may be O2 sensors and signal hypoxia by releasing ROS to cytosol and the oxidant signal may inhibit proline hydroxylases (making HIF-1-alpha stable so it can bind to HIF-1-beta)
        
        Preparation for oxidative stress or force theory - mechanisms used by animals to tolerate harsh environmental conditions that induce hypometabolic states
        
        Molecular oxygen is biradical (it has 2 unpaired electrons on its outer shell), reduction of one electron produces a superoxide ion, unpairing electrons makes oxygen very reactive
        
        Surviving Hypoxia
        
        There are two types of hypoxia survivors oxyconformers (conform to O2 level) and oxyregulators (maintain oxygen until they have to conform)
        
        Acute (short term) exposure is coped with by maintaining oxygen delivery to tissues by hyperventilating and maintaining or increasing cardiac output
        
        Prolonged exposure can be dealt by:
        
        Oxygen carriage optimised by modification in the concentration and affinity of extracellular respiratory pigment
        
        Hypometabolism, downregulation of protein synthesis and regulatory enzymes
        
        Eventually anaerobic metabolism has to happen
        
        Gene regulation: the transcription factor Hypoxia inducible factor -1 (HIF-1) receives signals from molecular oxygen sensors via a redox/phosphorylation reaction and then HIF-1-alpha (a subunit) activates the transcription of a number of genes involved in the hypoxia response this includes:
        
        Erythropoietin (EPO) increases red blood cell production in a process called erythropoiesis
        
        Vascular endothelial growth factor - enhances proliferation (amount) of blood vessels by angiogenesis (forming new blood vessels from pre-existing blood vessels to increase O2 supply
        
        Anaerobic enzymes - upregulated to enhance anaerobic metabolism by enolase (which enhances glycolysis and glucose uptake)
        
        Transcription factors - downregulates unnecessary processes to save energy
        
        Glucose transporters - upregulated to increase glucose transport to enhance anaerobic metabolism
        
        HIF1
        
        DNA-binding heterodimeric (2 different molecules) transcription factor, composed of an alpha (unstable, degrades rapidly in normoxia so when it is present there can be a quick response to hypoxia) and beta subunit
        
        Low oxygen: alpha subunit is going to bind to the beta subunit and a series of other molecules that will allow it to function, in this case there is no hydroxylation so HIF beta binds then transcription factor p300 as it isn't blocked
        
        High oxygen: a series of mechanisms will inhibit transcription and target it for degradation, proline and asparagine are amino acids that are residues in the alpha subunit and when oxygen is present they undergo hydroxylation (adding -OH) by enzymes (proline hydroxylases (causes VHL proteolysis) and factor inhibiting HIF(blocks p300)) because of this transcription factor p300 is blocked so alpha subunit binding to the beta subunit is inhibited so HIF transcription activity doesn't happen, hydroxylation also encourages the protein VHL to bind, VHL modifies other proteins by binding them with ubiquitin which labels them for degradation
- - - - Some key things to remember about phylogenetics are that there are phylogenetic hotspots, as for marine specific factors, there are less barriers and a higher dispersal chance
  - - - Cladogram - represents relative recency of common ancestry (branch lengths don'tt represent time)
      - Phylogram - represents the same as a cladogram but with an indication of branch length indicating the amount of evolutionary change over time
      - Polytomy - when you don't have enough info to determine the branching order
      - Methods for creating phylogenetic trees
        
        Genetic distance method: calculate genetic distance = number of differences between two taxa to see the evolutionary change over time. Normally Nei's (Dn) genetic distance, where the different bases are counted, assumes that after species diverge from their ancestor that they will differ even more, multiple substitutions may happen
        
        Neighbour Joining (NJ): builds tree based off distance, uses an algorithm, calculations involve manipulating the distance matrix (a table that shows the different points in space), for each pair of taxa the distance between the two aligned sequences is recorded in the matrix, a tree is then made from the matrixes
        
        Pros: Fast, can give you an overview
        
        Cons: Underestimates the amount of change, not reliable when dealing with deeper divergence times, not accepted for publication
        
        Maximum Parsimony (MP): Parsimony is based off the idea that the tree makes as little changes possible, based off characters (bases) only and not invariant characters (nucleotides), algorithm determines the least amount of steps necessary for the tree to have that agrees with the data, this process does need a consensus (general agreement) tree
        
        Pros: Mid-range/ average computing cost, routinely accepted for publication
        
        Cons: Fails to take into account many factors of sequence evolution (reversals, convergence/homoplasy (similar features in different lineages)), not reliable when dealing with deeper divergence times
        
        Maximum Likelihood: Makes an evolutionary tree by finding a tree that is most likely correct based off the data, character based so applies to each site (bases), searches for trees that are consistent with the model and the data
        
        Pros: Can apply a model of sequence evolution, preferred by reviewers
        
        Cons: Computationally expensive, can get trapped when it searches foor trees as it considers one tree only once
        
        Bayesian Analysis (BA): It looks at the most likely trees then summarises it (taking into account probability and evolution) to best explain data, it is character based (bases), doesn't require bootstrapping as it already calculates reliability (0.9 is reliable)
        
        Pros: Applies it to evolution sequence, preferred by reviewers, considers the same tree many times so it doesn't get trapped
        
        Cons: Expensive
        
        Bootstrapping: is a method used after Neighbourhood Joining, Maximum Parsimony and Maximum Likelihood to calculate the reliability by making trees using the same methods and comparing it, many replicates can be made and for every clade present in the original and the replicate is given a value of 1, 0.7 is statistically acceptable, it can take a while but even longer for ML (hours or even days)
        
        Marker choice is selected based off the question, as the resolution varies of each marker, on top of that different markers need to be picked if the question is interspecific or intraspecific
        
        If the genetic closeness is at species level mtDNA is used and ITS rDNA
        
        If the genetic closeness is from genera to families large or small sub unit rDNA is used as well as mtDNA
        
        If the genetic closeness is at class to phyla large or small sub-unit of rDNA is used
        
        Calibrating phylogenetic trees with molecular clocks
        
        By looking at events in time that we know of we can measure the rate of change of DNA sequences through time, genes tend to have a higher synonymous substitution (doesn't alter protein) rate than non-synonymous substitution (alters protein), though rates of non-synonymous substitution vary among genes.
        
        Rates of change vary across taxa and body size as small bodies are associated with small life spans which means that more generations will happen in the same amount of time resulting in more change
    - - Phylogenetic signals describe the degree to which more closely related organisms share more similar traits. But with some species they have symbionts associated with it, sometimes there are variations of spots near the species name, the larger the spot the more diverse the microbial community
      - Long branch attraction is when the amount of change accumulated within a lineage is sufficient to cause that lineage to appear similar to another long lineage branch and they have both changed a lot and not because they are related by descent
      - Missing data and taxon sampling can be due to a lot of human error, it is important to consider what you can actually conclude from your work taking into account the errors
- - - - Maximising numbers, protecting genetic diversity and hybridising subspecies all present the risk of outbreeding depression (the crossing of genetically differentiated populations can lead to reduced population fitness (less reproductive success))
      - Maximising genotypic diversity, local adaptation and keeping subspecies separate has the risk of inbreeding depression (inbreeding has deleterious effects that result in reduced population fitness (survival rate decreases))
      - Broadcast spawners (species that release gametes into the water) make it hard to work out what gamete belongs to who, lots of populatation can be made but from limited parents
    - - Success is measured by analysing who the parents are by genetic techniques to see what genes are passed on, family roles also needed to be respected, if species were monogamous in the wild they were also monogamous ex-situ
      - Microsatellites were used to overcome the challenges when it came to breeding broadcast spawners
      - Hybrid detection
      - Even after release a couple of generations later the genes can be tested
    - - To breed animals that are threatened with extinction so they don't become extinct
      - To also conserve genetic diversity not just the quantity
      - Aims in ex-situ (out of their normal environment) breeding
        
        Preventing extinction of species that don't have an immediate chance of survival
        
        Establish sources for founding new populations in the wild
        
        Provide demographic (numbers) and genetic support for wild populations (these conflict with eachother)
  - - - To start DNA of the threatened species is extracted and amplified to see the remaining haplotypes, study the species breeding habits eg are they monogamous, and check the parentage of the offspring to see how many parents need to sustain the species, from there captive breeding could be an option but nonetheless a decision will be made
    - - Genetic diversity needs to be protected in MPAs as it gives them a chance to adapt to change and to evolve, to monitor genetic diversity microsatellite data is collected from species in MPAs and outside MPAs to make sure living in MPAs isn't reducing genetic diversity, many small MPAs face challenges of inbreeding however they are more common as they are easier to manage. It is best to increase the number and distribution of MPAs as there is evidence of environmental recovery
    - - Commercially caught fish tend to mature earlier and are smaller due to fishery induced evolution, to maintain genetic diversity part of the population isn't harvested so wild stock is maintained to introduce to harvested areas so genetic impacts are reduced