BARCODING TERRESTRIAL INVERTEBRATES

Julie K. Stahlhut

Biodiversity Institute of Ontario

The ideal specimen

• High resolution image• (MANDATORY for free processing at

CCDB!)

• Remainder of specimen kept as voucher

• Less than 15-20 years old

• We have sequenced pinned museum specimens > 50 years old.• Lower success rate• Most sequences not full-length

Pinned and dried

≥ 95% ethanol ≤ -20ºC

Before putting tissue into a plate ....

• Static electricity can make dry arthropod legs “jump”!

• Add ethanol to each well first.

How much tissue should I add to each well?

• Minute invertebrate:• Whole body*

• Small arthropod: • Whole leg/antenna (5-6

mm)

• Large arthropod: • Tibia or femur (2-4 mm)

• Soft invertebrate: • Small tissue fragment • (2 mm3)

* Voucher recovery possible

Avoiding contamination

• Keep work surfaces clean.

• Sanitize dissecting tools between specimens.

• Consider a PCR hood if you do your own molecular work.

PROBLEM: Specimen age

• DNA degrades over time.

• Standard primers often fail on older specimens.

Solutions

• Choose fresher material whenever possible.

• Don’t mix old and fresh specimens on same plate.

• Use mini-primers on old specimens.

© 2011 Iowa State University – Osborn Research Club

PROBLEM: Multiple higher taxa in one project

• Most primers have broad applicability.

• But some primer-taxon combinations work better than others.

Solutions

• Separate plate for each higher taxon.

• Or -- check primer lists to find best match to all of your specimens.

Group First choice primers

Spiders C_LepFolF + C_LepFolR

Bees LepF1 + LepR1

Lice LCO1490_t1 + HCO2198_t1

Mosquitoes C_LepFolF + C_LepFolR

PROBLEM: DNA degradation

• Some collecting and storage methods are not DNA friendly.

Solutions

• Collect in cyanide jars or ≥95% ethanol.

• Empty water traps at least every 2-3 days.

• Refresh ethanol and store vials at ≤ -20ºC.

• Keep pinned material dry and cool.

• Propylene glycol? Mixed results.

Ethyl acetate

Wet traps Heat and humidity

Formalin

PROBLEM: Non-target amplification

Solutions

• Follow best pinning and preserving practice

• Mold, bacteria

• Cross-contamination • Sterile plate-loading technique• Watch out for butterfly scales, Drosophila cultures, etc.

• Use the most specific primers possible

• Avoid abdominal tissue

• Wolbachia• Common endosymbiont of

arthropods and nematodes

• Pseudogenes/numts • Use good templates• Check BOLD sequence trees

“I did everything right but my sequences looked wrong!”

• Invertebrates are extremely diverse!

• Sometimes one subtaxon doesn’t work well with suggested primers.

• Indels are common in haplodiploids (Hymenoptera, thrips)

• Experiment with lab methods

• Experiment with alignment methods

• Contact us

Acknowledgements

• Dario Lijtmaer• Rodolphe Rougerie• Gerry Blagoev• Jeff Webb• Alex Wild • Suz Bateson• The folks at BugGuide.net