Anopheles gambiae:

A genetic approach

Karin EiglmeierUnité de Biochimie et Biologie Moléculaire des Insectes

Institut Pasteur

MosquitoesOrder : Diptera

Toxorhynchitinae

Anophelinae Culicinae

Family: Culicidae

Aedes CulexAnopheles

Since when .......????

Anopheline fossile (20 Myr)

http://www.uky.edu/AS/Geology/webdogs/amber/critters/skeeter-b.html

(Amber from the Dominican Republic)Dr. David Grimaldi

Cretaceous carnivorous dinosaures (145-65Myr)

Oldest Culicidae-like fossile: 76-79 Myr

(canadian amber)

FROM: www.dinozaury.ovh.org/ index.php?id=galeria

Generalized mosquito life cycle

The larvae feed on microorganisms and organic matter in the water. 4 -14 days

The pupal stage:non-feeding stage of development, reacts to stimuli-> metamorphosis1- 4 days

Eggs hatch into larvaewithin 48 H.

Larvae

Pupa

Eggs

Adult

modified from: www.edmonton.cal

Mosquito facts

CO2, temperature, humidity, odor, colour (infra-red) mouvement

MalariaWest Nile virusfilarial diseasesdengue encephalitisyellow fever

1 -1.5 miles/hour 75 - 100 miles20 - 35 meters

Detection Transmission

Immunity

Blood-meal

endophage4x weight

Mosquito facts

CO2, temperature, humidity, odor, colour (infra-red) mouvement

MalariaWest Nile virusfilarial diseasesdengue encephalitisyellow fever

1 -1.5 miles/hour 75 - 100 miles20 - 35 meters

Detection Transmission

Immunity

Blood-meal

endophage4x weight

Malaria (“Paludisme”)

- Vector : Anopheles gambiae

- Parasite : protozoans of the genus

Plasmodium

> 90 countries (40% of the world’s population)

- 90% malaria fatalities in sub-saharan Africa

- 300 - 500 millions clinical cases/ year

- 1.5 - 2.7 millions deaths

Plasmodium life cycle

Adapted from Waters, Science,301 (2003)

~13-18 days

Anopheles mosquitoes

Genus: Anopheles

Africa Asia America

An. gambiaeAn. arabiensisAn. funestus

An. stephensiAn. farautiAn. sinensisAn. tellessarusAn. minimus

An. albimanusAn. quadrimaculatusAn. darlingiAn. freeborni

Principal disease transmitting species:

about 70 transmit malaria to humans about 20 are important vectors

- An. gambiae M et S- An. arabiensis- An. melas- An. merus- An. bwambae- An. quadriannulatus- An. quadriannulatus B

Anopheles gambiae complex

Adapted from J.Mouchet & D.Fontenille

Golden Path length ~ 273 Mb

( from: http://www2.ncid.cdc.gov/vector/vector.html)

Anopheles gambiae chromosomes

Whole genome assembly

Genome size: - 278 Mb ( 273 Mb )

- 8987 scaffolds

- 303 scaffolds => 91% of the genome

- 18634 contigs

Inter-scaffold gaps: - sequence gaps, no clones

- repeat sequences

- smaller scaffolds ?

Y chromosome: - no assembly - ongoing ? 0.18Mb

- high repeat content

Current release AgamP3 last update: 2/2006First draft: March 2002

49 61 42 53 24 Mb

Anopheles gambiae chromosomes

+ +

adapted from: http://www.ensembl.org

Immediate results

20 genes (1999) 14700 genes (2005)

Cross disciplinary research:- Drosophila community- Bioinformaticiens- other domains- BLAST

Fieldwork:- Entomologists- Identification follow-up of mutations

adapt strategy

Gene prediction and annotation

Several evidences: - Gene prediction programs:

- open reading frame- signals: start codon, stop codon, poly-

adenylation site - splice sites- bias in base composition- bias in base frequency

- encoded peptide has similarity with known protein

- encoded peptide has similarity with a protein domain or motif

- « evolutionarily conserved sequences » - Ecores

- cDNA, EST, SAGE biological evidences

Annotation

15189 genes

Celera pipeline (Otto) Ensembl pipeline

“Ab inito gene finding” Homology

9896 transcripts14564 transcripts

24460 transcripts

15189 genes

13757840 5974(identified exclusively) (identified exclusively)

“consensus set”

Does gene prediction correspond to real gene ?

Problems:- real gene? => mono-exonic !

- small exons, intron-exon structure

- first and last exon

- untranscribed regions (“UTRs”)

- genes for atypic or specific proteins

- genes duplicated in tandem

- pseudo-genes

Comparison An. gambiae - D.

melanogaster

Common ancestor

Anopheles gambiae Drosophila melanogaster

250

~ 273 Mb15189 annotated genes(13765 in AgamP3)

~ 130 Mb 14651 annotated genes

Protein similarity

14000

12000

10000

8000

6000

4000

2000

Dm Ag

44,2%

11,0%

15,9%

10,3%

18,6%

47,2%

13,8%

17,9%

10,0%

11,1%species specific

Homologs, best matches: - non-insects

Homologs, best matches: - insects

“Many-to-many” orthologs,duplications

1:1 Orthologs (6089 pairs)average identity: 56%

13885 12981

increased speed of divergence :

Orthologs Human - Fugu:average identity: 61%

adapted from: Zdobnov E. Science (2002), 298, 149-159

Starting point: Publication of the Anopheles gambiae genome 2002

- Genome sequence incomplete

- First characterisation and annotation of genes of variable quality

Problems: Post-genomic analysis difficult

Gene detection

Approach: Full-length enriched cDNA libraries:

- developmental stages

- different tissues

Aims: - Identification of new genes

- Improve description of gene structure (TSS, UTRs, Exon / Intron)

- Alternative splicing recombinant proteins

- Facilitate comparative genomics

Genome, gene expression and annotation

How to get more

information?

How to get more

information?

Experimental evidence:

- Transcriptome

- Proteome

- Biochemistry

- RNAi

- Transgenesis

Modified from Zhang MQ Nat. Rev. Genet.2002(9):698-709 and from Ben-Dor,S

1 2 3 4 5

ATG STOP Poly(A) site

TTSTSS

Promoter

1 2 3 4 5

ATG STOP Poly(A) site

DNA

Pre-mRNA

mRNA 1 3 42

AUG

5

STOP

Poly(A)

5’UTR 3’UTRCDS

CAP

From DNA to mRNA

Modified from Zhang MQ Nat. Rev. Genet.2002(9):698-709 and from Ben-Dor,S

1 2 3 4 5

ATG STOP Poly(A) site

TTSTSS

Promoter

1 2 3 4 5

ATG STOP Poly(A) site

DNA

Pre-mRNA

mRNA 1 3 42

AUG

5

STOP

Poly(A)

5’UTR 3’UTRCDS

CAP

From DNA to mRNA

AAAAAAAAAAAA

Gpppp

OH

mRNA + CAPmRNA sans CAPmRNA sans CAP

AAAAAAAAAAAA

GpppOH

OH

AAAAAAAAAAAA

pOH

OH

AAAAAAAAAAAA

OHOH

BAP treatment

TAP treatment

RNA ligation5’-oligo

« Oligo-capping » ( Maruyama & Sugano )

adapted from:Suzuki et al. Genome Res.(2001)11(5):677-84

AAAATTTT

TTTT

TTTTAAAA

TTTTAAAA

Ligation

First strand synthesis

Alkaline degradation

PCR

SfiI Digestion

TTTT

AAAA

adapted from:Suzuki et al. Genome Res.(2001)11(5):677-84

Banques ADNc « Full-length »

modified from: www.edmonton.cal

More genes to discover ?

67044 reads

~ 3700 clusters

85 % improved

cDNAs - submitted to EMBL :

654 new genes

Pilot project: Adult females

3032 Ensembl genes

Perfect annotation

Gene modelcDNA dataproteins

- Ensembl -

Improvement of annotation

- Ensembl -

600 new genes

- how are they?

”New” genes

- Ensembl -

Predictions and proof

- Ensembl -

Banques ADNc « Full-length »

Clustering results:5664 cluster - 869 new genes

175

21

6

12

256

37

401

Adulte females(4056)

Embryos(1816)

Larvae(1982)

Available or planned cDNA libraries:

Available/sequenced:

• Adult females

• Embryo

• Larves

• Salivary glands

Planned:

• Pupa

Plasmodium life cycle

Adapted from Waters, Science,301 (2003) Adapted from: James, A.A., (2003),206:3817-3821

Sporozoites- invasion, specific receptors- secretory cells- storage- influence normal functions

Saliva- proteins involved in

l’hematophagy- modulation of immune defense

An. gambiae salivary glands

Chromosome X, pos. 11070 kb: Anopheles-specific SG1 family

AgamP3

Moz2a v.34Adapted from: Arca et al. J.ExpBiol(2005), 208:3971

What does genomics offer for malaria control ?

Adapted from Waters, Science,301 (2003)

Vector eradication

Vector eradication

Insecticides

• Monitoring of insecticide resistance genes - pyrethroid resistance

- epidemiology

• Detoxifying enzymes • Detox-chip

• New targets

Adapted from Waters, Science,301 (2003)

Host - vector relationship

Host - vector relationship

Behaviour Candidate genes:

odorant - smell 79 putative odorant receptorsgustatory - taste 76 putative gustatory receptors

Attractant/Repellant - Host location

Vectorial capacity

Mating

Oviposition Traps

Adapted from Waters, Science,301 (2003)

Transmission blocking - SM1

Transmission blocking - SM1

SM1 = salivary gland and midgut peptide 1 (Gosh, et al. 2000)

12 amino acids

transgenic mosquitoes:

- midgut expression - blocking plasmodium

- salivary gland expression - blocking invasion

- selective advantage of transgenic mosquitoes

- proof of principle => Marrelli et al. , PNAS 104, 2007

Adapted from Waters, Science,301 (2003)

Immune system

Immune system

Natural efficient immune system

- resistance against Plasmodium

- melanotic incapsulation - several loci

- identify genes

- multiply/release naturally occuring resistant strains

Adapted from Waters, Science,301 (2003)

Salivary glands

Salivary glands

- cDNAs

- RT-PCR

- protein expression

- SAGE analysis of transcriptome

- proteomic studies of salivary glands and saliva

salivary gland sporozoite relationships

- saliva and humans:

- immunomodulatory

- function?

- vaccine target?

Where to go from now ??

Annotation = continuing process

- new start for Anopheles research

- Improvement of genomic annotation:

- Sequence, gene models, promoters

- genome arrays :

-infected non-infected

- Annotation at the protein level:

- protein interaction networks

- hypothesis experiments

- comparative genomics - more insects (honey

bee, Aedes)

- transgenic mosquitoes , RNAi

experiments

Aedes aegypti

- Start : September 2002

- Fin : Spring 2007 ?

first assembly, version 0.5

- Public:

- First annotations

- cDNA sequences

- Genome size: > 1300 Mb => in 4758 supercontigs

- « Evolutionary distance » Anopheles - Aedes:

~ 27-62 Myr

From: www.bg-sentinel.com

Gene size comparisonAnopheles

Aedes

Gene size comparison4 kb

16 kb

Anopheles

Aedes

Aedes aegypti annotation

- genome size bigger than expected (780 Mb => 1300 Mb)

- sequencing strategy different

- cDNAs early in project

- high content of repeat sequences (~68%)

- gene prediction programs adapted

- long genes, nested genes

- Anopheles - Aedes

- synteny between chromosome arms

Data bases

EnsemblAnopheles gambiae

Aedes aegypti

AnobaseAnopheles gambiae

VectorbaseAnopheles gambiae

Aedes aegyptiIxodes scrapularis

TIGR - The Institut for Genome Research

Traps

Identification

Understanding- behaviour

Genome/proteomecaracterisation

Promoter/expression

Vector control strategies: - Transgenesis

Genetically modified mosquitoes-reduced parasite transmission-functional genomics

New insecticidal targets

Vaccines

Main orientations in mosquito research

Immunity

Collaborations

Institut Pasteur

BBMI - Paul BreyCharles RothInge HolmPierre Dehoux (PF4)Shawn GomezSylvie PerrotMarie-Kim ChaverocheJean Sautereau

Plate-forme Genomique - PF1Christiane BouchierAnthony Lepelletier

Genoscope

Jean Weissenbach

Beatrice SegurensPatrick WinckerGabor GyapayCorinne da SilvaBetina Porcel

AMSUD Network

Sergio Verjovski-AlmeidaUniversidade de Sao Paulo

Hamza el DorrySuely L. GomesCarlos F.M. MenckAna L. Nascimento