Collecting, preparing, and labeling specimens Taxonomic Workshop for Early Detection of Important Tortricidae and Other Lepidopteran Agricultural and Silvicultural Pests
UMass Amherst 15-17 July 2013
Todd M. Gilligan, Ph.D. Colorado State University Department of Bioagricultural Sciences and Pest Management 1177 Campus Delivery Fort Collins, Colorado 80523 USA [email protected] / [email protected]
Collecting, preparing, and labeling
Collecting Lepidoptera ◦ Blacklighting ◦ Pheromone trapping ◦ Baiting ◦ Aerial netting ◦ Rearing Killing agents Labeling specimens Storing and curating Lepidoptera Handling moths on sticky traps Collecting Lepidoptera
Blacklight with sheet or trap – most general collecting Pheromone trapping – usually sticky traps, targeted species Baiting – adult-feeding species Aerial nets – diurnal species (butterflies) Rearing – targeted species Fogging – diversity studies Blacklighting
Ultra-violet blacklight ◦ BLB or BL tubes ◦ Typically 8-24 watts ◦ Approx. 370 nm wavelength ◦ Can run off standard 12V car batteries Mercury vapor ◦ Typically 160-1000 watts ◦ Approx. 350-375 nm wavelength ◦ Need a generator to run in the field Blacklighting – sheet
Ultra-violet blacklight White sheet Mercury vapor light optional Blacklighting – sheet Blacklighting – sheet
Collecting specimens ◦ Killing jars Kills specimens (may need to relax later) More later… ◦ Vials Keep specimens alive (possibly for few days) Preferred for microlepidoptera (including tortridids) ◦ Hypodermic needle Can inject larger specimens with ethanol or another killing agent (Sphingidae, Saturniidae) Kills quickly, specimens need relaxed later Blacklighting – traps
Ultra-violet blacklight Bucket trap ◦ Vanes / light ◦ Funnel ◦ Rain-drain ◦ Killing agent ◦ Power source / batteries Can leave unattended overnight
Blacklighting – traps
www.leptraps.com www.bioquip.com Blacklighting – traps
Considerations when placing a trap (or light and sheet) ◦ Place traps along flyways (forest edges, ridge lines, streams, natural high points) ◦ Avoid cold air pockets (inversions) ◦ Maximize light distribution – don’t place trap in dense forest ◦ Lights probably only attract moths from approx. 6 meters away ◦ Bucket traps are only 40% efficient at best
Blacklighting – traps
Egg cartons keep beetles from destroying trap
White bags = ammonium carbonate (killing agent) Pheromone trapping
Synthetic pheromones available for many economically important groups ◦ Sesiidae, Tortricidae, Erebidae (Lymantriinae)
“wing trap” “moth catcher” “delta trap” Pheromone trapping
Trap selection (for pest species) The “delta trap” and “wing trap” (both sticky traps) are usually the most sensitive trap to use for monitoring these moths. However, the “moth catcher trap” may be used in dusty conditions or in high moth population density. Do not re-use traps to monitor different insects as this may lead to mixed catches!
Trap density Two traps per hectare (2 traps/ha) for small holdings and in fields with uneven topography. One trap for every two hectares in large scale fields of homogenous lands.
Trap position Place trap near the highest point of the plant using supporting posts approximately 1 meter high, or higher if the crop is higher.
Trap data and interpretation Collect data weekly from the start of the flight of the over wintering generation. During the height of the population more frequent reading may be needed. Decisions on pesticide application should not be taken solely on the trap catch data. Climatic and biological considerations should be taken in account. Pheromone sticky traps
“Delta traps” commonly used for collecting pest species – single sticky board Sticky traps
Handling and shipping sticky traps
A & B: open and unfold trap C: place 2-3 packing peanuts in area of trap with no moths D: fold trap, secure with rubber band, place in plastic bag
Sticky traps
Specimens can also be cut individually or in small groups out of traps and stored in small plastic jars Sticky trap removal
Miller, R. S., S. Passoa, R. D. Waltz & V. Mastro. 1993. Insect removal from stick traps using a citrus oil solvent. Entomological News. 104(4): 209‐213. Sticky stuff = polyisobutylene (PIB) Citrus oil = Histoclear Wear gloves, don’t contaminate dissecting equipment Sticky trap removal
Create temporary label to track specimen through process Cut piece of sticky trap containing specimen or remove abdomen from specimen Immerse sticky trap piece or abdomen in Histoclear until specimen is free of PIB (floats free of trap) Soak briefly in clean Histoclear if necessary Remove specimen or abdomen and place in petri dish with Kimwipe or paper towel to dry
Removal of all (or most) PIB will result in a much easier and cleaner dissection Repeat several rinses in Histoclear rinses with abdomen if performing dissection Sticky trap removal New “hard-tack” or “no-mess adhesive” sticky traps Less mess (can handle without gloves) Effective (according to OTIS lab) Specimens must still be removed with Histoclear or citrus oil AlphaScents inserts Baiting
Many species do not come to lights (especially females) ◦ Collect live females for eggs/rearing Types of baiting ◦ Sugaring – paint tree trunks ◦ Sugar rope – wine + sugar solution on ropes ◦ Cylinder mesh traps Basic ingredients ◦ Sugar/molasses, yeast, rum/beer, rotten fruit Baiting
www.leptraps.com Aerial netting (butterfly net)
Used by most collectors to sample butterflies and diurnal macromoths Some microlepidoptera (including tortricids!) do not come to light and are only collected during the day
Hystrichophora loricana – Gilligan et al. 2009 Rearing
Rearing from immature stages ◦ Eggs, larvae, pupae ◦ Associate larvae with adults ◦ Obtain biological data ◦ Document pests / verify hosts In many cases, the only way to positively identify intercepted pest species Rearing
Microlepidoptera (including Tortricidae) can be easy to rear ◦ Search for larval damage ◦ Remove leaves/fruit and place in rearing container or plastic zip-lock bag “condiment cups” are ideal for leaf-rolling tortricids ◦ Change or add leaves/food as needed ◦ Avoid mold, spiders, bacterial infection Rearing Killing agents
Various (and some very dangerous!) killing agents are regularly used ◦ Cyanide ◦ Ethyl acetate ◦ Ammonium hydroxide ◦ Ammonium carbonate Freezing is a safe alternative (although not ideal for microlepidoptera) I use and recommend ammonium carbonate for tortricids Killing agents
potassium cyanide ethyl acetate ammonia
For collecting at sheet For placement in trap
Killing agents
Ammonium carbonate
◦ (NH4)2CO3 ◦ “Bakers ammonia” ◦ Leavening agent for baking bread, primary ingredient in smelling salts ◦ Breaks down into ammonia, carbon dixoide, and water ◦ Relatively safe for humans (much safer than most other killing agents) ◦ Keeps specimens “relaxed” for an extended period for ease of spreading Ammonia killing jar
Plastic peanut butter jar with a sock containing powdered ammonium carbonate Freezing specimens
When no other killing agent is available Microlepidoptera dehydrate quickly and need to be relaxed to spread Macrolepidoptera can often be thawed and spread after freezing Pinning and spreading moths
Optional demonstration on Wednesday or during collecting trip Labeling specimens
Collection data is as important as the specimen! Proper format for labels is important Absolute minimum: locality and date Other information that should be included: ◦ Collector, GPS coordinates, elevation, method of collecting Appropriate size (< 1.5 cm X 1.0 cm) ◦ 4 point font standard Use quality, archival paper and printing ◦ Acid-free, 100% rag (cotton) ◦ Archival printing (India ink, laser printing, NOT inkjet!) Labeling specimens
USA: AL: Baldwin Co. Weeks Bay Estuarine Reserve Pitcher Plant Bog boardwalk KENYA: Kakamega District N 30° 24.971’ W 087° 49.144’ Kakamega Forest Reserve 21 Jun 2008, el. 43’ UV light Rondo Retreat Centre, rainforest Gilligan, Wright & Gibson colls. N 00°13’37.9” E 034°53’04.6” 23-26 Nov 2010, el. 1598m T. Gilligan & A. Mukiri colls. UV AUSTRALIA: QLD
6 Victor Place, Kuranda S 16º48.842', E 145º38.611' el. 393m, 13 Dec 2009 T. & M. Gilligan, M. Moulds
Storing and curating Lepidoptera
The most important consideration for specimen storage is prevent damage from other insects (dermestid beetles, silverfish, booklice, etc.) Best stored in drawers with tight-fitting lids Collection boxes are okay as long as fumigated regularly Store specimens in a cool, dry place ◦ Especially important for DNA extraction at a later time – humidity (water) destroys DNA! Storing and curating Lepidoptera
Organizing collections is personal preference ◦ Can be organized taxonomically, but taxonomy often changes ◦ USNM tortricids are grouped by subfamily and tribe, and then arranged alphabetically by genus ◦ Other families are organized by current phylogenetic relationships Most important to curate collection regularly regardless of organization ◦ Specimens will otherwise become lost ◦ Dermestid damage will become a large problem if undetected ◦ Data will be lost unless specimens are labeled promptly and properly Storing and curating Lepidoptera