Presented by M. Virgilio Royal Museum for Central Africa, Tervuren, Belgium Royal Belgian Institute...

presented by

M. VirgilioRoyal Museum for Central Africa, Tervuren, Belgium

Royal Belgian Institute of Natural Sciences, Brussels, Belgium

[email protected]

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

Tephritid Barcoding Initiative

and barcoding of agricultural pest


Why DNA Barcode Fruit Flies?

• large number of species

• many economically relevant species

• difficult identification of larvae.

• need for standardized diagnostic methods


The Tephritid Barcode Initiative (TBI)

TBI Chair:Bruce McPheron, Penn State

TBI Coordinators:Allen Norrbom, USDA, USAMarc De Meyer, RMCA, Belgium

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 ZealandNorman Barr, USAAmnon Freidberg, IsraelHo-Yeon Han, South KoreaGeorge Roderick, USAIan White, UK


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


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


Who is generating fruit fly barcodes?

1. Penn State University, USA: Bruce McPheron, Md. Sajedul Islam

2. Lincoln University, New Zealand: Karen Armstrong

3. Royal Museum Central Africa, BE: Marc De Meyer, Massi Virgilio

4. Yonsei University, Korea: Ho-Yeon Han

5. California Department of Agriculture, USA: Peter Kerr

6. Smithsonian National Museum of Natural History, USA: Allen Norrbom

7. APHIS-PPQ Mission lab, USA: Norman Barr

8. University of Guelph

9. Biodiversity Institute of Ontario


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


What needs to be provided in BOLD for TBI

Euleia fratria (Trypetinae)TEPH101 (from BOLD)

1. identification of specimen by an expert

taxonomist

2. voucher specimen

3. collection information (collection date

and location)

4. other infos (GPS, elevation,

photodocumentation) not mandatory but

strongly encouraged

5. barcode: at least 500bp with less than

1% missing data.

6. trace files stored in BOLD.

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

http://www.boldsystems.org/views/taxbrowser.php?taxon=Trypetinae

Deposited in :

Tephritidae {family}

Subfamilies (5)Dacinae [572] Phytalmiinae [7] Tachiniscinae [1] Tephritinae [219] Trypetinae [243]

Lineage: Arthropoda: Insecta: Diptera

Specimen Record: 1047

Specimens with barcodes: 828

Public Sequences: 240

Search performed on October 20, 2008

http://www.barcodinglife.org

Barcodes : Species :

213

73

533

95

240

44 34

119

19 37

0

200

400

600

An

astr

eph

a

Rh

ago

leti

s

Bac

tro

cera

Cer

atit

is

Dac

us

all species EI species

Five Genera of Highest Economic Importance

0

20

40

60

80

100

An

astr

eph

a

Rh

ago

leti

s

Bac

tro

cera

Cer

atit

is

Dac

us

number of species % of species barcoded

insights into

DNA barcoding

of tephritid fruitflies


0%

4%

8%

12%

16%

20%

Bactrocera Ceratitis Dacus

between species

within species

intra- and inter-specific genetic differentiation in 3 genera of tephritid fruitflies

p-distances

12.6 6.3 6.6

ratio inter/intra specific p-dist


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

K2p = 0.02% (S.E.= 0.01%)

average genetic distance

within species

Ceratitis fasciventris

C. anonae

C. rosa

... and where not:

the FAR complex

0.00

0.01

0.02

withinspecies

betweenspeciesK

2P

average genetic distances

ratio = 1.2

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)

Dacus:

from identification issues

to classification issues

as expected low genetic differentiation

in taxonomically closer species...

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

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

COINJT-K2P

DACUS

DIDACUS

LOPHODACUS

LEPTOXYDA

NEODACUS

PSILODACUS

...but also discrepancies between morphological and molecular taxonomy

subgeneric classification

Apocynaceae

Cucurbitaceae

Passifloraceaeunknown

COI+16S+perBayesian

DNA barcoding may provide clues for an improved morphological classification

host plant choice

DNA barcoding may reveal cryptic speciation:

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


methodological problems

in the barcoding of tephritid fruitflies

from museum collections


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.


in the barcoding of museum specimens

age of specimens vs barcoding success

% specimens amplified

% specimens sequenced



0

20

40

60

80

100

2007

2006

2005

2004

2003

2002

2001

2000 90

s

80s

< 1

980

(n=394)

0

20

40

60

80

100

2007

2006

2005

2004

2003

2002

2001

2000

90s

80s

< 1

980

from EtOH specimens

from pinned specimens

% of succesfully sequenced specimens (n=394)



pinned vs EtOH preserved specimens

>200041%

90s4%

80s3%

70s1%

60s2%

50s13%

40s4%

<194032%Ceratitis, Bactrocera and Dacus

in the collections of the RMCA:(n=1804)



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.





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




the BARFLY projectshort 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



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.

% of pcr products

obtained

% of barcode sequences

obtained (>500bp)

(n=229)

0

20

40

60

80

100

>2000 90s 80s 70s 60s 50s 40s <1940

fragment 1

fragment 2

fragment 3

.

(n=229)

>2000 90s 80s 70s 60s 50s 40s <1940

0

20

40

60

80

100

higher performances

compared to the standard primers

0

20

40

60

80

100

>2000 90s 80s <1980

standard primers

internal primers

(n=229)

+7% +32% +6% +7%


% of barcodes obtained (>500bp)

RMCA:

internal primers as a standard protocol

for the barcoding of museum specimens

collected before 2000




work in progress:

could ‘minibarcodes’ represent a

temporary alternative to

‘full’ barcodes?


Dacus armatus

400 bp

600 bp

500 bp

400 bp

400 bp


300 bp

400 bp

500 bp

Bactrocera oleae


TBI current challenges

– Coordination / tracking specimens: BOLD

– Older specimens: mini barcodes / internal primers

– Fresh material: collecting activities

– Taxon coverage: non EI genera

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: [email protected]

Massimiliano Virgilio: [email protected]:

RMCA current challenges

RMCA is opened to collaborations 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: [email protected]

Massimiliano Virgilio: [email protected]:


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)

Presented by M. Virgilio Royal Museum for Central Africa, Tervuren, Belgium Royal Belgian Institute...

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