L32 Experimental Evidence for Natural Selection

Question 1

Selectionist view

Answer 1

• most new mutations are deleterious - eliminated or maintained at low frequency
• vast majority of polymorphisms are stable, being maintained by balancing selection

Question 2

Neutralist view

Answer 2

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

Question 3

Demonstrating neutrality

Answer 3

• impossible to demonstrate absence of selection

- attempt to show amount and pattern of variation in genes within and between species fits the neutral model

Question 4

DDT

Answer 4

• first used as an insecticide

honey bees abandoning hives and dying due to insecticide use

Question 5

Insecticide action

Answer 5

see onenote

monogenic responses

metabolic resistance
target-site modification

Question 6

Step 1 - genetic variation must be shown to exist

Answer 6

To show that selection operates

Question 7

Resistance to everything

Answer 7

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

Question 8

Resistance to everything

Answer 8

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

Question 9

Over-expression of Cyp6g1 in transgenic flies

Answer 9

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

Question 10

Overexpression of Cyp6g1 caused by…

Answer 10

an insertion mutation

492 bp insertion at 5’ of the gene = LTR (long terminal repeat) of retrovirus called ACCORD

Question 11

Accord

Answer 11

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

Question 12

Tissue specificity of Cyp6g1 over-expression

Answer 12

see onenote

Brain = target for insecticide

Fat body, malpighian tubules and midgut - had role to metabolise the insecticide before it gets to the brain

Question 13

Further evolution at the Cyp6g1 locus

Answer 13

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

Question 14

Step 2 - allele frequencies must be described over time in one population or through space across populations

Answer 14

see onenote

Temporal and geographical changes in Cyp6g1 haplotype frequencies

Africa - no evidence of evolutionary form

Where insecticides are used => more elaborate evolutionary forms

Question 15

Step 3 - demonstrate phenotypic diversity among genotypes for an aspect of molecular functions

Answer 15

see onenote

Cyp6g1 metabolizes DDT

When cyp6g1 is expressed => DDT can be metabolised into DDD

Question 16

Following imidacloprid metabolism in vivo

Answer 16

see onenote

Question 17

Twin ion mass spectrometry

Answer 17

see onenote

Question 18

Twin ion mass spectrometry

Answer 18

see onenote

Question 19

Controlled over-transcription of Cyp6g1

Answer 19

see onenote

Accord driver-GAL4 x UAS-Cyp6g1

Question 20

Metabolized by Cyp6g1 and rapidly excreted

Answer 20

see onenote

higher levels of metabolism in flies overexpressing Cyp6g1

Question 21

Metabolised by Cyp6g1 but NOT detoxified

Answer 21

see onenote

Question 22

Step 4

Answer 22

knowing:

1. function of gene product
2. nature of functional differences between the products of the alleles
3. ecology of the organism

Postulate a relevant, discriminatory selective agent

Question 23

Selective agents

Answer 23

• 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

Question 24

Step 5 - Differences in molecular function must be reflected in fitness differences in postulated selective regime

Answer 24

see onenote

DDT correlates with Cyp6g1 haplotype class

The more elaborate the changes are to cyp6g1, the more resistant they are to DDT

Question 25

The Wiggle Index

Answer 25

see onenote

Question 26

Step 6

Answer 26

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

Question 27

Population association study

Answer 27

see onenote

Question 28

Does Cyp6g1-BP contribute to DDT resistance in natural population?

Answer 28

Normal distribution as a response to insecticide

• Proportion of individuals that carry Cyp6g1-BP haplotype, most derived haplotype
• Survived haplotype far more common

Question 29

Step 7

Answer 29

test hypothesis by perturbing allele frequencies in natural population