200 million humans infected (83 million with S. mansoni)Annual mortality 280,000 (130,000 due to S. mansoni)

Morbidity 120 million humans600 million more humans at risk

Impact of schistosomiasis second only to malaria (WHO)

SCHISTOSOMIASIS

TRANSMISSION?• Only 1-6% of snails shed cercaria

(transmit schistosomes)• Up to 50% of snails may be infected

with schistosomes (Hamburger et al, 2004)

• Experimental exposure of field-collected snails with low parasite doses does not always result in infection. High parasite doses yield ~100% infection

Why does Biomphalaria not always host S. mansoni? • Genetic determinants (Newton 1950; Richards and Shade, 1987)

• Differential expression/activity of defenses (Bender et al, 2007)

• Matching or mismatching phenotypes of recognition/ antigens of individual snails and parasites (Theron and Coustau, 2005).

Need more insight into snail internal defenses, compatibility

FLUKES AND SNAILS

parasites are excellent cell biologistsspecific manipulation of host cell function

Trichobilharzia ocellata

Schistosoma mansoni

Echinostomaparaensei

Adema et al., 1994a,bLoker & Adema, 1995 Adema et al., 1997a

SNAIL ANTI-PARASITE RESPONSE INCLUDES FREPs

FREP = FIBRINOGEN-RELATED PROTEIN

80-120

180-200

65

kDa

UNIQUE COMBINATION

IgSFS/ICRFBGC CN C

IMMUNOGLOBULIN SUPERFAMILY

(IgSF)-LIKE DOMAIN

FIBRINOGEN /b g (FBG)-LIKE DOMAIN

N C C C C C

CELL ADHERENCEMULTIMERIZATION

LECTIN FUNCTION NON-SELF RECOGNITION

FREP POLYMORPHISME H

Bg

MULTIPLE FBG- HYBRIDIZING SEQUENCES IN THE GENOME OF BIOMPHALARIA GLABRATA (Bg)

E= ECO RI, H= HAE III

BgMF2BgMF3

HtrF1

Balf4

BgSF1

BgMF4

BgMF1Balf1

Balf2Balf3

HtrF2

HtrF3

HtrF6

HtrF7 BtrF1

Balf5

BgBF1 BgRF1

Sea CucumberMouse

Human

LfllAb

LflAc

LflAa

OTHER PLANORBIDS ALSO HAVE FREP GENES.INTERSPERSED SEQUENCES FROM DIFFERENTSPECIES UNDERLINE POLYMORPHISMS

Biomphalaria glabrataBiomphalaria alexandrina

Bulinus truncatusHelisoma trivolvis

NJ TREE (AA)FREP FBG

DEUTEROSTOME FBG

SLUG FBG

QUESTIONS RE: FREPs • How are FREPs diversified, but not other genes?

• Do biological data agree with the in silico model?

• Genomic architecture of FREP loci?

• Regulation of FREP expression?

LOOK AWAY FROM THE LAMP POST

What don’t we know?

Acquired resistance

AMPsROIs

Hemolin (MDM)

NO productionFREPs

Toll

RNAi

DSCAM

A GAP IN MOLECULAR DATA

El-Sayed, Bartholomeu,Ivens, Johnston,LoVerde

Fist draft stage of 270Mb genome

COMPLETE 3.2 Gb

http://www.genome.gov/Images/dir_image.jpg

Snail Sympathizers &Molluscan Partisans

Informal group meeting

All are welcomeMonday, 2 July, from 1-2pm, in room C

ASP 2001

DEFENSE FACTORS• Non self recognition: beta-1,3-glucan binding protein,

selectin, galectin, hemolectin, C-type lectins, ficolin, FREPs, lipopolysacaharide protein-binding protein, peptidoglycan recognition protein

• Signal transduction/regulatory pathways: TNF-R, IFN-R SF, NILT-1, NFKB inhibitor, NFKB-like, Toll-like receptor, death-associated protein kinase, apoptosis inhibitor, arginine kinase, kinase-like protein, rabconnectin 3 b, transducin, proteases, serine protease inhibitors, transcription factors

• AMP: DRAGP, LBP-BPI, achacin, aplysianin, cystatins, theromacin

• Cytoskeleton/Cell adhesion: Actin, calponin, Myosin, amiloride binding protein, apical early endosomal glycoprotein, intermediate filament protein A, paramyosin, tropomyosin, tubulin, twitchin, supervillin, collagen, peroxinectin, Cadherin, hepatocyte growth factor, integrin

• Cell-mediated cytotoxicity/Lysosomal enzymes: carboxypeptidase A, cathepsin F, cellulase, disintegrin-like and metalloprotease, endo-1,4-mannanase, endo-1,4 glucanase, endochitinase, enolase, hydrolase, perforin aminopeptidase 2, Peptidase, macrophage expressed protein, vacuolar proton-translocating ATPase, peroxidase, peroxidases, amoebocyte agglutination factor

• RNAi: Helicase, PIWI/argonaute-like• Stress response proteins: several HSPs, SOD, GST

THE MITOCHONDRIAL GENOME OF Biomphalaria glabrata

DeJong, Emery, Adema, 2004 Journal of Parasitology 90:991-997

                                                                                                        Biomphalaria glabrata snails were collected by Dr Omar dos Santos Carvalho in the metropolitan region of Belo Horizonte, district of Barreiro, Brazil (19oS59min/44oW02min). Susceptible to Schistosoma mansoni

GENOME SEQUENCE TARGETBB02 STRAIN Biomphalaria glabrata

BAC-ACCESS TO GENOME

pAGIBAC1

61,824 BAC clones, average insert 136 Kb, 9.05x coverage of genome (931 Mb),

161 (384 well) plates

x pAGIBAC

GENOME “BAC-DOOR” ACCESS61,824 BAC clones161 (384 well) platesaverage insert 136 Kb 9.1x genome coverage

Target recoveredActin 45hsp70 13Myoglobin 22FREP4 4FREP13 7L32 15Ferritin 2Lectin 3Ubiquitin 3MnSOD 10

Clone Bg-BB 117G16 BAC end sequenceheat shock protein 70 [Biomphalaria glabrata] Length=636 Score = 229 bits (583), Expect = 3e-59 Identities = 110/123 (89%), Positives = 117/123 (95%), Gaps = 0/123 (0%) Frame = +3Query 12 MPGRNKAPAIGIDLGTTYSCVGVFQHGKVEIIANDQGNRTTPSYVAFTDTERLVGDAAKN 191 M GRNKAPAIGIDLGTTYSCVGVFQHGKVEIIANDQGNRTTPSYVAFTDTERLVGDAAKN Sbjct 1 MSGRNKAPAIGIDLGTTYSCVGVFQHGKVEIIANDQGNRTTPSYVAFTDTERLVGDAAKN 60

Query 192 QAAMNPSNTIFDAKRLIGRKFNDKTVQSXXKHWPFKVVEVDGKPKIKAEYRGEQKLFAPX 371 QAAMNPSNT++DAKRLIGRKF+DKTVQ+ KHWPFKVVEVDG+PKI+AEYRGE KLFAP Sbjct 61 QAAMNPSNTVYDAKRLIGRKFDDKTVQNDMKHWPFKVVEVDGRPKIQAEYRGENKLFAPE 120

Snail Genome Meeting November 6, 2007

Biomphalaria glabrata(Photo Courtesy of Matty Knight)

• Initial sequencing plan

• Revised sequencing plan

• Project direct costs

• Accessing data

• Project contacts

• High repeat content

• High heterozygosity levels

• High GC or AT content

• Large genome size

What are the properties of a difficult genome ?

Newbler Assembly 4.1 Input Data

Biomphalaria glabrata Final Improved Assembly Statistics Version 4.3

Sin

gle

IG

FR

EP

Du

al I

G F

RE

P

Biomphalariaglabrata

Aplysiacalifornica