vector biology & genetics Flashcards
1
Q
Anopheles distribution
A
- tip of africa to northern europe
- doesn’t match malaria distribution
- some species better at transmitting
- temperature and vector suitability determines malaria
- much higher in sub-saharan africa
- some suitable areas have no malaria
- mainly through prevention and healthcare
2
Q
EIR
A
- entomological inoculation rate
- how many infectious bites individual receives per unit time
- areas of high EIR
- hundreds of bites a season, each bite has 20-30% chance of transmission
- overlaps with Anopheles gambiae distribution
- A. gambiae - 90% of malaria-related deaths
3
Q
mosquitoes as vectors
A
- anthropophagic
- high parasite susceptibility
- lifespan long enough for parasite life cycle
- density and habitat preference
- ensures vertebrate host contact
- frequent blood feeding
- endophagic and endophilic
- eating and resting
4
Q
genetic approaches to studying vector biology
A
- chromosomal view
- classical genetic view
- genomics view
5
Q
species complex
A
- group of closely related species that fulfill the definition of a species, but are morphologically identical
- vary in:
- spatial or temporal association with each other
- physiological behaviour
- vector competence
- chromosomal and molecular markers
- used to study mating and gene flow
6
Q
A. gambiae complex
A
- 6 species in complex
- occupy different regions but are sometimes sympatric
- ss - sensu stricto
- sl - sensu lato
7
Q
biological species concept
A
- offspring of two separate species that have interbred will be sterile
- e.g. A. gambiae and A. arabiensis
- species in the complex fulfill this definition
- reproductively isolated groups
- species in the complex fulfill this definition
8
Q
chromosomal forms
A
- refers to particular class of chromosomes in mosquito cells that have high energy requirements and turnover
- salivary glands, ovaries, nerve cells
- variations in these chromosomes
9
Q
mosquito chromosomes
A
- 3 homologous chromosomes
- includes 1 sex chromosome
- cells with chromosomal forms have multiple rounds of replication without cell division
- forms polytene chromosomes
10
Q
polytene chromosomes
A
- multiple copies of chromosomes align to form one huge chromosome
- observed under light microscope
- characteristic banding pattern
- light and dark
- highly specific and reproducible
- corresponds to gene order
11
Q
chromosome inversions
A
- easy to identify with polytene chromosomes
- genetic markers in A. gambiae populations
- pericentric inversion
- includes centromere
- paracentric inversion
- doesn’t include cetnromere
- inversion requires 2 chromosome breaks and 180 degree rotation
12
Q
heterozygous inversion
A
- only 1 chromosome inverted
- lack of homology in inverted region
- can’t pair up properly
- inverted region loops out so homologous regions can bind
- characteristic markers
13
Q
frequency of inversions
A
- varies
- at least 5 types known
- some have been assoicated with adaptation to new ecological niches
- can use to study gene flow among populations
14
Q
inversion frequency in Mali
A
- sample transects of whole country
- 3 groups found upon genotyping
- different combinations of inversions
- Savannah group
- similar based on inversion class
- rain dependent larval sites
- Mopti group
- semi-permanent sites, humidity and water
15
Q
effect of inversions
A
- no loss/gain of genetic material
- generally no phenotypic effect
- in heterozygotes
- reproductive consequences
- due to homologue pairing at meiosis
16
Q
inversions and meiosis
A
- usually homologous chromosomes align and crossover occurs
- crossover can occur within inverted region
- loss of genetic information
- one dicentric gamete (2 centromeres)
- one acentric gamete
- neither are viable
- viable gametes only when no crossover
- also crossover suppression
- occurs at a reduced rate
17
Q
allele combinations at inversion genes
A
- very tight linkage
- allele combinations at several genes with inversion stay together for longer
- combinations may iprove adaptation to certain niches
- more likely to stay in that niche
- coadapted gene complex
- important in phenotypes that require several genes
- behavioural traits
18
Q
inversions in Savannah and Mopti
A
- tight linkage of different 2R chromosome inversion arrangements in these 2 groups with larval habitat preferences
- suggests inversions contain coadapted gene complexes
19
Q
identifying polytene chromosomes
A
- labour intensive in high numbers of mosquitoes
- → PCR-based assays
- amplify amplicon specific to one of two inversions (M or S)
- high throughput probing of west african mosquitoes
- some areas predominantly M, others S
- different relative abundances in some areas
- sympatric here
20
Q
M/S heterozygotes
A
- virtually non-existent in wild populations despite overlap
- heterozygotes are viable in lab
- must be something upstream of mating acting as a reproductive barrier
- pre-mating reproductive isolation
21
Q
speciation of M and S
A
- at the early stage of speciation
- will eventually form a separate species
- almost fulfill biological species concept but can occasionally mate to produce viable offspring
- S → A. gambiae ss
- M → A. coluzzi
22
Q
kdr
A
- knockdown resistance allele
- pyrethroid resistance
- originated and spread in S form
- sympatric M forms took some itme to acquire it
- then rapidly spread through M
- gene flow is restricted
23
Q
mosquito genome
A
- completed 2002
- 250 Mb, 15000 genes
- most sequences assembled onto chromsomes 2, 3 and X
- 1000 mosquito genomes project
- african mosquitoes sequenced to analyse gene flow and variation in isolated populations
- 16 genomes project
- evolution rates among different species
24
Q
genetics view
A
- focus on linkage analysis using marker genes
- small genetic markers ditributed among chromosomes
- track which regions go to which individuals in offspring
- which regions associated with certain phenotypes
25
genetic markers
* microsatellite repeats and SNPs
* common, neutral, dispersed
* high resolution mapping
* \>1 SNP per Kb ov Anopheles genome
* microsatellite repeats
* di/trinucleotides repeated multiple times
* varies among individuals
* identify with simple PCR
26
linkage analysis
* if an organism has obtained resistance:
* some individuals have a variant conferring this that is found in a certain chromosome
* = quantitative trait loci
* different markers around that chromosome can vary between indiviudals
* combination of markers = haplotype
* cross individuals of different haplotypes
* random combinations produced
* markers consistently associated with resistance allele are physically much closer
* use to identify allele location
* more markers = higher resolution
* SNPs also give higher resolution
27
anopheles resistance phenotype mapping
* isolation of 3 genomic regions with differential association with capacity to melanise parasites
28
microsatellite distribution to identify resistance island
* chromosome region 2L
* wild caught mosquitoes with wild falciparum
* microsatellites identified 15 Mb region
* regions annotated as immune genes or as being upregulated during falciparum infection → candidate genes
* 2 identified
* APL1 and 2
29
APL1/2
* anopheles plasmodium-responsive leucine rich repeat 1 and 2
* RNAi in infection experiments
* only APL1 has an effect on oocyst number in rodent malaria
* but may have an effect on natural transmission
30
genomics view
* involves e.g. microarrays
* cheap, simple
* spot gene probes with different coloured fluorophores onto plate
* expose to RNA from infected and non-infected mosquito
* hybridisation → fluorescence
* differential expression identified by different colours
* merge of 2 colours = both expressed
31
RNAi
* use in genome wide screening
* target a gene to silence
* observe change in phenotype
* only a transient change
32
CRISPR/cas9
* easy, works in most organisms
* cas9 nculease cuts to remove target
* knockout by NHEJ or insertion by homologous recombination
* may not always produce knockout
* e.g. 3bp deletion has no frame shift, may not knockout
