vector biology & genetics

Question 1

Anopheles distribution

Answer 1

• 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

Question 2

EIR

Answer 2

• 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

Question 3

mosquitoes as vectors

Answer 3

• 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

Question 4

genetic approaches to studying vector biology

Answer 4

• chromosomal view
• classical genetic view
• genomics view

Question 5

species complex

Answer 5

• 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

Question 6

A. gambiae complex

Answer 6

• 6 species in complex
• occupy different regions but are sometimes sympatric
• ss - sensu stricto
• sl - sensu lato

Question 7

biological species concept

Answer 7

• 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

Question 8

chromosomal forms

Answer 8

• 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

Question 9

mosquito chromosomes

Answer 9

• 3 homologous chromosomes
• includes 1 sex chromosome
• cells with chromosomal forms have multiple rounds of replication without cell division
  
  • forms polytene chromosomes

Question 10

polytene chromosomes

Answer 10

• 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

Question 11

chromosome inversions

Answer 11

• 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

Question 12

heterozygous inversion

Answer 12

• 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

Question 13

frequency of inversions

Answer 13

• varies
• at least 5 types known
• some have been assoicated with adaptation to new ecological niches
• can use to study gene flow among populations

Question 14

inversion frequency in Mali

Answer 14

• 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

Question 15

effect of inversions

Answer 15

• no loss/gain of genetic material
• generally no phenotypic effect
• in heterozygotes
  
  • reproductive consequences
  • due to homologue pairing at meiosis

Question 16

inversions and meiosis

Answer 16

• 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

Question 17

allele combinations at inversion genes

Answer 17

• 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

Question 18

inversions in Savannah and Mopti

Answer 18

• tight linkage of different 2R chromosome inversion arrangements in these 2 groups with larval habitat preferences
• suggests inversions contain coadapted gene complexes

Question 19

identifying polytene chromosomes

Answer 19

• 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

Question 20

M/S heterozygotes

Answer 20

• 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

Question 21

speciation of M and S

Answer 21

• 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

Question 22

kdr

Answer 22

• 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

Question 23

mosquito genome

Answer 23

• 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

Question 24

genetics view

Answer 24

• 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

Question 25

genetic markers

Answer 25

• 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

Question 26

linkage analysis

Answer 26

• 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

Question 27

anopheles resistance phenotype mapping

Answer 27

• isolation of 3 genomic regions with differential association with capacity to melanise parasites

Question 28

microsatellite distribution to identify resistance island

Answer 28

• 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

Question 29

APL1/2

Answer 29

• 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

Question 30

genomics view

Answer 30

• 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

Question 31

RNAi

Answer 31

• use in genome wide screening
• target a gene to silence
• observe change in phenotype
• only a transient change

Question 32

CRISPR/cas9

Answer 32

• 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