DNA-barcoding on a different scale: challenges within

Coccoidea

Lyn Cook, The University of Queensland, Australia

SIBIScale Insect Barcode Initiative

(Stellenbosch, Feb 2007)

Outline

• What are scale insects?

• Why have they been a challenge?

• Prospects for a universal scale

insect barcode

APHIDS SCALE INSECTS

10 MY210 - 160 Myr ago

1st scale insect “barcode”• 1995 - DNA extracted from new

morphotype. Some ovarian tissue

frozen at -70C. Cuticle slide mounted as

voucher. Other specimens from same

collection frozen or stored in 100%

ethanol

• 1999 - PCR and sequencing

• 2003 - paper published including

sequences and stored DNA as part of

holotype material

Apiomorpha gullanae

A. munita

A. munita18S rDNA (nuclear SSU rRNA)

18S variation within a morphospecies

PCR product for 5’ end of nuclear ssu rDNA (18S)

360 bp

480 bp

720 bp

980 bp1160 bp

*single genus

18S helix E10-1

18S NJ K2P+G

Cryptococcus

COI 5’

barcode

COI 3’ tRNA numts

*mealybugs few many + yes?

*armored scales

no many +/-

*soft scales no some

felt scales few some +/- yes?

*archaeococcoids

few few yes?

*stictococcoids no no

other scales no some* major pest groups

results of COI barcoding attempts

Why such low success with COI?

AT A C G T

COI 69-85 23-42 8-17 6-19 28-47

3rds ≤ 97.5

30-58 2-19 0-5 27-54

COII 72-86 28-52 8-23 4-15 23-43

3rds ≤ 100 38-65 0-26 0-16 22-55

mtDNA nucleotide frequencies (%)

COI barcode region

• Taxa that amplify appear to have higher CG

in 3rd positions than failures– not direct comparison

• Amino acid divergence of up to 8% in aphid

crown

• Amino acid divergence of up to 55% within

scale crown

18S var. 28SD2

var.

*mealybugs most many most most

*armored scales

most x most most?

*soft scales most most many

most

felt scales most most many

most

archaeococcoids

most most many

most

*stictococcoids most most

other scales most most yes in many

most

Alternatives

Florida lac scale

(= lobate lac scale)

Case study

Paratachardina lobata

28S

conserved regions only

COI - 3’ region

28S D2 region for lac scales

length of the region varies from 703-1114

28S D2

Aligned using

muscle -no

adjustment

Analysed using NJ

K2P

India

India

Florida

Christmas Is.

Bermuda

&, as of 8 weeks ago, Cuba

Conclusions• Proceed with non-COI barcode - 28S when successful

• 18S might be an alternative in “decision tree”

-18S to assign specimen to group or direct ID

- next step depends on grouping (e.g. COI barcode or COI 3’, 12S etc with group-specific

primers)

• Attempt to find solution to COI barcode problem. Options include:

• more primer design

• mini COI barcode

• cocktails

• whole mt genome

• other?

SIBI, Stellenbosch, February, 2007Participants: BB Normark, W Pieterse, PJ Gullan, I Millar, J Giliomee, D Downie, F Haas, C Eardley, LG Cook, A Rung, D Masinga, A Venter, D Muller, DR Miller, R Hanna

Acknowledgements

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