BARCODINGTERRESTRIALINVERTEBRATESJulie K. StahlhutBiodiversity Institute of Ontario
The ideal specimen • High resolution image • (MANDATORY for free processing at CCDB!) Pinned and dried • Remainder of specimen kept as voucher • Less than 15-20 years old • We have sequenced pinned museum specimens > 50 years old.≥ 95% ethanol • Lower success rate ≤ -20ºC • Most sequences not full-length
SAMPLING PROCESS: PLATE PREPARATION Before putting tissue into a plate ....PLING PROCESS: PLATE PREPARATION To begin the sampling process, position the Co o begin the sampling process, position the Row Markers A01 Column Markers • Static electricitythe platecanA01 plate on a flat surface with label make dry Row Markers ate on a flat surface with the plate label facing towards you. A12acing towards you. arthropod legs “jump”! he column markers (1–12) should (1–12) should be at The column markers be at • Add ethanol to each the top and the row markers (A–H) should well first.he top and the row markers (A–H) should H12e on the left side. be on the left side. IMPORTANT! 30 l or 1 drop Add Ethanol IMPORTANT! 30 l to avoid static or 1 cross-contamination Add Ethanol samples are prone to spontaneous displacement because of static to avoid static ectricity (e.g., arthropod legs), sampling wells should be pre-filled with cross-contamination0 l (microlitres) of 95-100% Ethanol, e.g., using a multi channel pipettor. a pipettor samples are prone to spontaneoustodisplacement because of static If is not available, add one drop of Ethanol each well using electricity (e.g., arthropod legs), sampling wells should be pre-filled withn eyedropper, just prior to sampling.ote: Do not ladd excess ethanol - this may cause well caps to pop off during shipping. If the 30 (microlitres) of 95-100% Ethanol, e.g., using a multi channel pipettor.amples If a compact and not available, fixed one drop of Ethanol to each welltissue), are pipettor is were previously add in ethanol (e.g. moist vertebrate muscle using en adding fixative to the plate is optional. an eyedropper, just prior to sampling.
Note: If using bleach or deterge tools by thoroughly rinsing them degradation.How much tissue should I add to each well? Below are some examples of r• Minute invertebrate: • Sm • Whole body* • La• Small arthropod: • Ve • Whole leg/antenna (5-6 • 2-d mm) • Mi• Large arthropod: Note: D • Tibia or femur (2-4 mm) extracti• Soft invertebrate: fragmen • Small tissue fragment Avoid s • (2 mm3) Avoid s with dig * Voucher recovery possible To visualise well contents (e.g., to amount of fixative or tissue sa
Avoiding contamination• Keep work surfaces clean.• Sanitize dissecting tools between specimens.• Consider a PCR hood if you do your own molecular work.
PROBLEM: Multiple higher Solutions taxa in one project • Separate plate for each• Most primers have broad higher taxon. applicability.• But some primer-taxon • Or -- check primer lists to combinations work find best match to all of better than others. 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 Solutions degradation• Some collecting and storage methods are not DNA • Collect in cyanide jars or friendly. ≥95% ethanol. • Empty water traps at least every 2-3 days. • Refresh ethanol and Formalin store vials at ≤ -20ºC.Ethyl acetate • Keep pinned material dry and cool. • Propylene glycol? Mixed Wet traps results. Heat and humidity
PROBLEM: Non-target Solutions amplification• Mold, bacteria • Follow best pinning and preserving practice • Sterile plate-loading technique• Cross-contamination • Watch out for butterfly scales, Drosophila cultures, etc.• Wolbachia • Use the most specific • Common endosymbiont of primers possible arthropods and nematodes • Avoid abdominal tissue• Pseudogenes/numts • Use good templates • Check BOLD sequence trees
“I did everything right but my sequences looked wrong!”• Invertebrates are • Experiment with lab extremely diverse! methods• Sometimes one • Experiment with subtaxon doesn’t work alignment methods well with suggested primers. • Contact us• Indels are common in haplodiploids (Hymenoptera, thrips)
Acknowledgements• Dario Lijtmaer• Rodolphe Rougerie• Gerry Blagoev• Jeff Webb• Alex Wild• Suz Bateson• The folks at BugGuide.net