L32 Experimental Evidence for Natural Selection Flashcards
Selectionist view
- most new mutations are deleterious - eliminated or maintained at low frequency
- vast majority of polymorphisms are stable, being maintained by balancing selection
Neutralist view
Vast majority of polymorphisms:
- s = 0
- different genotypes have same relative fitness (selective neutrality)
- polymorphisms transiet
- only important factors u and Ne
- selection is neutral, does not discriminate
Demonstrating neutrality
- impossible to demonstrate absence of selection
- attempt to show amount and pattern of variation in genes within and between species fits the neutral model
DDT
- first used as an insecticide
honey bees abandoning hives and dying due to insecticide use
Insecticide action
see onenote
monogenic responses
metabolic resistance
target-site modification
Step 1 - genetic variation must be shown to exist
To show that selection operates
Resistance to everything
see onenote
1961 - Kikkawa mapped dominant DDT resistance locus to 64.5cM in Hikone R strain of Drosophila melanogaster
resistance to multiple insecticides, maps to a region that contains the cytochrome P450 gene, Cyp6g1
In all eukaryotes cytochrome P450 responsible for drug metabolism
Resistance to everything
see onenote
1961 - Kikkawa mapped dominant DDT resistance locus to 64.5cM in Hikone R strain of Drosophila melanogaster
resistance to multiple insecticides, maps to a region that contains the cytochrome P450 gene, Cyp6g1
In all eukaryotes cytochrome P450 responsible for drug metabolism
expression of cyp6g1 higher in DDT resistant lines in comparison to susceptible lines
Over-expression of Cyp6g1 in transgenic flies
see onenote
heat shock driver-GAL4 x UAS-Cyp6g1
The only difference to susceptible and resistant flies is that Cyp6g1 is overexpressed in Cyp6g1
Resistance to multiple insecticides in conferred by overexpression of Cyp6g1 gene
Overexpression of Cyp6g1 caused by…
an insertion mutation
492 bp insertion at 5’ of the gene = LTR (long terminal repeat) of retrovirus called ACCORD
Accord
see onenote
contains cis-regulatory elements
accord LTR drives expression of GFP in midgut, malpighian tubules and fat bodies when placed in front of minimal promoter
- Malpighian tubules = human kidney
- Fat bodies = human liver
- Good enzyme in the perfect place to metabolise insecticide
Liver and kidney are both filtering organs
Tissue specificity of Cyp6g1 over-expression
see onenote
Brain = target for insecticide
Fat body, malpighian tubules and midgut - had role to metabolise the insecticide before it gets to the brain
Further evolution at the Cyp6g1 locus
see onenote
Cyp6g1 duplicated
Insertion of other transposons
None of these evolutions were seen in older stocks, began when DDT started being used in the field, also had an increase in cyp6g1 expression
Step 2 - allele frequencies must be described over time in one population or through space across populations
see onenote
Temporal and geographical changes in Cyp6g1 haplotype frequencies
Africa - no evidence of evolutionary form
Where insecticides are used => more elaborate evolutionary forms
Step 3 - demonstrate phenotypic diversity among genotypes for an aspect of molecular functions
see onenote
Cyp6g1 metabolizes DDT
When cyp6g1 is expressed => DDT can be metabolised into DDD
Following imidacloprid metabolism in vivo
see onenote
Twin ion mass spectrometry
see onenote
Twin ion mass spectrometry
see onenote
Controlled over-transcription of Cyp6g1
see onenote
Accord driver-GAL4 x UAS-Cyp6g1
Metabolized by Cyp6g1 and rapidly excreted
see onenote
higher levels of metabolism in flies overexpressing Cyp6g1
Metabolised by Cyp6g1 but NOT detoxified
see onenote
Step 4
knowing:
- function of gene product
- nature of functional differences between the products of the alleles
- ecology of the organism
Postulate a relevant, discriminatory selective agent
Selective agents
- DDT postulated to be OG selective agent but then banned
- many different insecticides could now be selective agents
Many different insecticides now could be responsible for maintaining this polymorphism
Step 5 - Differences in molecular function must be reflected in fitness differences in postulated selective regime
see onenote
DDT correlates with Cyp6g1 haplotype class
The more elaborate the changes are to cyp6g1, the more resistant they are to DDT
The Wiggle Index
see onenote
Step 6
Natural population must be re-examined to seek a comprehensive explanation for observed allele frequency distributions in space and time
Not achieved:
- Drosophila is not a pest
- insecticides and concentrations exposed to in nature not known
Population association study
see onenote
Does Cyp6g1-BP contribute to DDT resistance in natural population?
Normal distribution as a response to insecticide
- Proportion of individuals that carry Cyp6g1-BP haplotype, most derived haplotype
- Survived haplotype far more common
Step 7
test hypothesis by perturbing allele frequencies in natural population
