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Tephritid Barcoding Initiative and barcoding of agricultural pest. presented by M. Virgilio Royal Museum for Central Africa, Tervuren, Belgium Royal Belgian Institute of Natural Sciences, Brussels, Belgium m.virgilio@skynet.be.

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slide1

Tephritid Barcoding Initiative

and barcoding of agricultural pest

presented by

M. Virgilio

Royal Museum for Central Africa, Tervuren, Belgium

Royal Belgian Institute of Natural Sciences, Brussels, Belgium

m.virgilio@skynet.be

Royal Museum for Central Africa – Royal Belgian Institute of Natural Sciences

slide2

Why DNA Barcode Fruit Flies?

  • large number of species
  • many economically relevant species
  • difficult identification of larvae.
  • need for standardized diagnostic methods

Royal Museum for Central Africa – Royal Belgian Institute of Natural Sciences

slide3

The Tephritid Barcode Initiative (TBI)

  • CBOL obtained funding from the Sloan Foundation
  • to support a “Demonstrator System”
    • Steering Committee formed in April, 2006, in Belgium

Steering Committee Members:

Karen Armstrong, New Zealand

Norman Barr, USA

Amnon Freidberg, Israel

Ho-Yeon Han, South Korea

George Roderick, USA

Ian White, UK

TBI Chair:

Bruce McPheron, Penn State

TBI Coordinators:

Allen Norrbom, USDA, USA

Marc De Meyer, RMCA, Belgium

Royal Museum for Central Africa – Royal Belgian Institute of Natural Sciences

slide4

TBI proposal: Goals

Generate barcode database for 2,000 species

  • 5 individuals/species (10,000 specimens)
  • 100% of economically important (EI) species
  • >75% of EI congeners
  • 1 species per genus in subtribes containing EI species
  • 1 species per genus in other higher taxa
  • representative tephritoid families

Royal Museum for Central Africa – Royal Belgian Institute of Natural Sciences

slide5

TBI proposal: Beneficial Outcomes

1) Establish a globally-available DNA database of barcodes

2) Establish a globally-available DNA repository

3) Generate a collection of identified vouchers for future systematic work

Royal Museum for Central Africa – Royal Belgian Institute of Natural Sciences

slide6

Who is generating fruit fly barcodes?

  • Penn State University, USA: Bruce McPheron, Md. Sajedul Islam
  • Lincoln University, New Zealand: Karen Armstrong
  • Royal Museum Central Africa, BE: Marc De Meyer, Massi Virgilio
  • Yonsei University, Korea: Ho-Yeon Han
  • California Department of Agriculture, USA: Peter Kerr
  • Smithsonian National Museum of Natural History, USA: Allen Norrbom
  • APHIS-PPQ Mission lab, USA: Norman Barr
  • University of Guelph
  • Biodiversity Institute of Ontario

Royal Museum for Central Africa – Royal Belgian Institute of Natural Sciences

slide7

Financial support

Penn State University, USA: technical support

APHIS: collecting & sequencing

Belgian Federal Government: sequencing, pilot study mini-barcodes

California Citrus Board: general expenditure

Korean Government: sequencing

(Dutch Government: collecting)

Various contributions through staff time and general core funding of institutions or other projects

Royal Museum for Central Africa – Royal Belgian Institute of Natural Sciences

slide8

What needs to be provided in BOLD for TBI

  • identification of specimen by an expert taxonomist
  • voucher specimen
  • collection information (collection date and location)
  • other infos (GPS, elevation, photodocumentation) not mandatorybut strongly encouraged
  • barcode: at least 500bp with less than 1% missing data.
  • trace files stored in BOLD.

Euleia fratria (Trypetinae)TEPH101 (from BOLD)

Other COI records (e.g., Genbank submissions) are integrated into the BOLD database but kept separate.

Royal Museum for Central Africa – Royal Belgian Institute of Natural Sciences

slide9

http://www.barcodinglife.org

Tephritidae {family}

Barcodes :

Species :

Search performed on October 20, 2008

Lineage: Arthropoda: Insecta: Diptera

Specimen Record: 1047

Specimens with barcodes: 828

Public Sequences: 240

Subfamilies (5)

Dacinae [572]

Phytalmiinae [7]

Tachiniscinae [1]

Tephritinae [219]

Trypetinae [243]

Deposited in :

slide10

all species

EI species

% of species barcoded

100

80

60

40

20

0

Dacus

Ceratitis

Rhagoletis

Bactrocera

Anastrepha

Five Genera of Highest Economic Importance

number of species

600

533

400

240

213

200

119

95

73

44

37

34

19

0

Dacus

Ceratitis

Rhagoletis

Bactrocera

Anastrepha

slide11

insights into

DNA barcoding

of tephritid fruitflies

Royal Museum for Central Africa – Royal Belgian Institute of Natural Sciences

slide12

intra- and inter-specific genetic differentiation

in 3 genera of tephritid fruitflies

ratio inter/intra specific p-dist

12.6 6.3 6.6

20%

16%

p-distances

12%

between species

8%

within species

4%

0%

Bactrocera

Ceratitis

Dacus

Royal Museum for Central Africa – Royal Belgian Institute of Natural Sciences

slide13

DNA barcoding of tephritid fruitflies:

where it works nicely....

44 barcodes of Bactrocera cucurbitae

from 11 countries:

  • Bangladesh
  • Cambodia
  • China
  • Hawaii
  • India
  • Kenya
  • La Reunion
  • Malaysia
  • Philippines
  • Sudan
  • Tanzania

average genetic distance

within species

K2p = 0.02%

(S.E.= 0.01%)

slide14

... and where not:

the FAR complex

Ceratitis fasciventris

C. anonae

C. rosa

average genetic distances

0.02

K2P

0.01

between

species

within

species

0.00

ratio = 1.2

slide15

other potentially “problematic”

tephritid species groups and complexes:

B. dorsalis complex

C. cosyra complex

C. capitata / C. caetrata

T. occipitale / T. quadrimaculatum

...

Armstrong and Ball (2005) Phil. Trans. R. Soc. B 360: 1813-1823. (B. dorsalis complex)

Virgilio et al. (2008) Molecular Phylogenetic and Evolution 48: 270-280 (FAR complex, Ceratitis)

slide16

Dacus:

from identification issues

to classification issues

as expected low genetic differentiation

in taxonomicallycloserspecies...

D. chiwira - D. famona, p-dist.= 0.3%(subgenus Dacus)

D. apostata - D. triater, p-dist.= 0.0% (subgenus Psilodacus)

slide17

DACUS

DIDACUS

LEPTOXYDA

LOPHODACUS

NEODACUS

PSILODACUS

COI

NJT-K2P

...but also discrepancies between

morphological and molecular taxonomy

subgeneric classification

slide18

Apocynaceae

Cucurbitaceae

Passifloraceae

unknown

DNA barcoding may provide clues

for an improved morphological classification

host plant choice

COI+16S+per

Bayesian

slide19

DNA barcoding may reveal cryptic speciation:

Virgilio M., De Meyer M, WhiteI.M., Backeljau T. (submitted) Phylogenetic relationships among African Dacus species (Diptera: Tephritidae) as inferred from mitochondrial and nuclear DNA.

slide20

methodological problems

in the barcoding of tephritid fruitflies

from museum collections

Royal Museum for Central Africa – Royal Belgian Institute of Natural Sciences

slide21

methodological problems

in the barcoding of museum specimens

DNA Extraction Procedures:

Several pilot studies performed to assess the success of an invasive, nondestructive DNA extraction method for museum specimens.

Study 1: L. Weigt and A. Driskell (Laboratory of Analytical Biology, National Museum of Natural History, Smithsonian Institution) -16S and COI

Study 2: N. Barr (USDA) and R. Ruiz (Penn State) -16S

Study 3. M. Virgilio (Royal Museum Central Africa, BE) – 16S, COI

Study 4. Md. Sajedul Islam (Penn State) - COI (ongoing study)

Qiagen DNeasy kit:

ok for DNA from recent dry and alcohol specimens not crushed during the extraction process.

Royal Museum for Central Africa – Royal Belgian Institute of Natural Sciences

slide22

100

% specimens amplified

80

% specimens sequenced

60

40

20

0

90s

80s

2007

2006

2005

2004

2003

2002

2001

2000

< 1980

methodological problems

in the barcoding of museum specimens

age of specimens vs barcoding success

(n=394)

slide23

100

from EtOH specimens

80

from pinned specimens

60

40

20

0

< 1980

2007

2006

2005

2004

2003

2002

2001

2000

90s

80s

methodological problems

in the barcoding of museum specimens

pinned vs EtOH preserved specimens

% of succesfully sequenced specimens (n=394)

slide24

<1940

>2000

32%

41%

40s

4%

50s

90s

80s

13%

70s

4%

3%

1%

60s

2%

methodological problems

in the barcoding of museum specimens

Ceratitis, Bactrocera and Dacus

in the collections of the RMCA:

(n=1804)

slide25

methodological problems

in the barcoding of museum specimens

DNA extraction protocols tested:

  • Qiagen DNeasy
  • Qiagen Biosprint
  • Bio101 Ancient DNA kit
  • Autogen
  • Machery-Nagel Filterservice kit
  • Phenol-Chloroform
  • DNAzol
  • E.Z.N.A. kits for forensics and insects
  • chargeswitch magnetic beads
  • etc.

Royal Museum for Central Africa – Royal Belgian Institute of Natural Sciences

slide26

methodological problems

in the barcoding of museum specimens

  • problem:
  • the quality of DNA rapidly decreases in time (shearing)
  • the amount of barcodes obtained from older museum specimens is not significantly affected by
    • the extraction method used
    • the use of genus- and species-specific primers
  • objective:
  • develop internal primers to improve the collection of barcodes from sheared DNA

Royal Museum for Central Africa – Royal Belgian Institute of Natural Sciences

slide27

methodological problems

in the barcoding of museum specimens

  • the BARFLY project
  • short term project funded by the Belgian Science Policy (BELSPO)
  • Joint Experimental Molecular Unit
  • of RMCA and RBINS (www.jemu.be)
  • Jeroen Van Houdt,Floris Breman
  • development of internal primers for the barcoding of Tephritids
  • collection of new barcodes from museum specimens

Royal Museum for Central Africa – Royal Belgian Institute of Natural Sciences

slide28

LCO 1490

HCO 2198

full barcode - c. 670 bp

frag. 1 - 343bp

frag. 2 - 269bp

frag. 3 - 227bp

a new set of internal primers

for the barcoding of tephritids

VanHoudt J., Breman F. C., Virgillio M.,De Meyer M. (in prep.)

A protocol for DNA barcoding of African tephritid fruitflies from museum collections using mini barcodes.

slide29

100

(n=229)

80

60

40

fragment 1

20

fragment 2

0

>2000

90s

80s

70s

60s

50s

40s

<1940

fragment 3

100

.

(n=229)

80

60

40

20

0

>2000

90s

80s

70s

60s

50s

40s

<1940

% of pcr products

obtained

% of barcode sequences

obtained (>500bp)

slide30

+7%

+32%

+6%

+7%

100

80

60

standard primers

40

internal primers

20

(n=229)

0

>2000

90s

80s

<1980

a new set of internal primers

for the barcoding of tephritids

higher performances

compared to the standard primers

% of barcodes obtained (>500bp)

slide31

a new set of internal primers

for the barcoding of tephritids

RMCA:

internal primers as a standard protocol for the barcoding of museum specimens collected before 2000

Royal Museum for Central Africa – Royal Belgian Institute of Natural Sciences

slide32

work in progress:

could ‘minibarcodes’represent a temporary alternative to

‘full’ barcodes?

Royal Museum for Central Africa – Royal Belgian Institute of Natural Sciences

slide33

Dacus armatus

400 bp

400 bp

500 bp

600 bp

400 bp

Royal Museum for Central Africa – Royal Belgian Institute of Natural Sciences

slide34

Bactrocera oleae

500 bp

300 bp

400 bp

Royal Museum for Central Africa – Royal Belgian Institute of Natural Sciences

slide35

TBI current challenges

  • Coordination / tracking specimens: BOLD
  • Older specimens: mini barcodes / internal primers
  • Fresh material: collecting activities
  • Taxon coverage: non EI genera

Royal Museum for Central Africa – Royal Belgian Institute of Natural Sciences

slide36

RMCA current challenges

RMCA is collecting barcodes as a “complementary activity” to the currently ongoing research lines:

  • phylogeny and population genetics of African Dacus
  • molecular taxonomy of species complexes in the genus Ceratitis
  • phylogeny of African and Australasian Bactrocera
  • population genetics and phylogeography of B. cucurbitae

Marc De Meyer: marc.de.meyer@africamuseum.be

Massimiliano Virgilio: m.virgilio@skynet.be

contact:

slide37

RMCA current challenges

RMCA is opened tocollaborations with African Institutions aiming to:

  • identify tephritid samples through morphological and molecular characters
  • prepare species inventories from African countries
  • collect / rear tephritid fruit flies
  • investigate population genetics and host races evolution of tephritid species

Marc De Meyer: marc.de.meyer@africamuseum.be

Massimiliano Virgilio: m.virgilio@skynet.be

contact:

slide38

Acknowledgments

Karen Armstrong

Abdelaziz Babikir

Thierry Backeljau

Norman Barr

Marc DeMeyer

Sajedul Islam

Bruce McPheron

researches at the RMCA are currently funded by the

Belgian Science Policy Action 1 (project MO/37/017)

and benefit from practical support by the

Joint Experimental Molecular Unit (JEMU)

Royal Museum for Central Africa – Royal Belgian Institute of Natural Sciences