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Wet preservation Following fixation, the most commonly used preservative is ethyl alcohol in various concentrations. Aphids and scale insects be prese...

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317331 Asst. Prof. Dr. Atiporn Saeung, Ph.D.

Objective After class, students are able to describe… - methods for collecting and preserving of insects

Collecting 1

Habitats and niches (breeding sites)

2

Timing and seasonality (diurnal, nocturnal/hot, rainy or dry season)

3

Biology and behaviour (life cycle/animal or human-biting insects)

4

Permit requirements (e.g. National Park)

Upton and Mantle (2010)

Breeding habitat

Anopheles spp. (larval stage)

Simuilum spp. (larval stage) Black flies (แมลงริ น้ ดำ)

Timing and seasonality

Black fly collection (adult female) during 7-9 a.m.

Biology Life cycle

Egg Adult

Larva (Nymph & naiad)

Pupa Mosquitoes

Behaviour Anthropophilic (human-biting mosquitoes)

Zoophilic (animal-biting mosquitoes) Original photograph from Dr. Petchaboon Poolphol, Office of Disease Prevention and Control No. 10th, Department of Disease Control, Ministry of Public Health, Ubon Ratchathani, Thailand

Collecting Methods and Equipment

 1. Land insects

Arial net: light nets for capture of insects in flight (flies, mosquitoes, butterflies, dragonflies, wasps, bees)

Sweep net: heavy, canvas bag with heavy mounting ring for net, large numbers of insects found in grass and plant foliage (small bugs, beetles, spiders)

Arial net

Sweep net

 Aspirator: small insects (mosquitoes, biting midges, black flies) Glass tubing

Glass tubing

Gauze cover

Rubber tubing

Glass mouthpiece

Mouth aspirator with HEPA filter

Glass vial aspirator

 Sifting soil and leaf litter: spring tail, beetles

Sifter

 Berlese (Tullgren) funnel (extraction device): mites

 Trap: adult insects Flight-interception traps: malaise trap and window trap

Malaise trap (e.g. black flies)

Window trap: heavy bodied insects (e.g. beetles)

Upton and Mantle (2010)

Sticky trap: bugs, bees, flies

Bait trap: blow flies

Klong-klaew et al. (2017) Sontigun et al. (2018)

Sanit et al. (2018)

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Baited pitfall trap: ground-dwelling insects (e.g. ground beetles)

https://en.wikipedia.org/wiki/Pitfall_trap

Light trap and sheet: night-flying insects

Upton and Mantle (2010)

Light traps (with CO2): mosquitoes, biting midges, sand flies

Miniature CDC Light Trap with UV Light

CDC mini light trap with incandescent light ttps://www.bioquipinc.com

 2. Aquatic insects  Hands, forceps: larvae/pupae of black flies

 Dip net: larvae of mosquitoes, mayflies, naiad of dragonfly

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 White (plastic) tray: larvae of mosquitoes

• Pipette/sucking tube: larvae of mosquitoes

Killing Methods  Freezer : small moths, butterflies (kill immediately prior to pinning and spreading)

 Killing bottle/tube: adult stage of most insects  Size of the bottle depend on the type of insect  Wide-mounted and made of glass (polypropylene or polythene jars can be used)  Clean bottle and avoiding from direct sunlight  Awareness of delicate specimen  Labeled POISON and keep out of reach of children  Handled with great care and read the chemical MSDS

Killing bottle Cork

Absorbent paper Cotton wool/sawdust Plaster & killing agent (e.g. ethyl acetate)

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Use of liquid for killing and fixing  70-95% ethyl alcohol  Hood’s solution (70-80% ethyl alcohol, glycerin)

1

2

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- 95% ethyl alcohol - distilled water - formaldehyde - glacial acetic acid

- 95% ethyl alcohol - distilled water - 40% formalin - glacial acetic acid

- 80% ethyl alcohol - formaldehyde - glacial acetic acid - picric acid

Kahle’s solution

Pampel’s solution

Alcoholic Bouin’s solution Millar et al. (2000)

Larvae/nymph (naiad)  70-80C hot water (e.g. mosquito larvae)  70-95% ethyl alcohol  KAAD mixture - 95% ethyl alcohol - kerosene - glacial acetic acid - dioxane  XA mixture - 95% ethyl alcohol - xylene

Preservation  Temporary storage

 Envelope: butterflies, dragonflies, stone flies

 Permanent storage

 Dry preservation  Wet preservation  Microscope slide

 Dry preservation  Relaxing methods: dishes, boxes, Barber’s relaxing fluid

Barber’s fluid - 95% ethanol - Distilled water - Ethyl acetate - Benzene

Millar et al. (2000)

 Pinning insects - entomological pins - forceps

Pinning blocks

Pin positioning for 8 orders

Wing arrangement for pinning insects

https://www.qm.qld.gov.au/microsites/wild/pdf/ preserving-pinning-insects-handout.pdf

Mounting beetle on a card platform

Millar et al. (2000)

Pinning adults in Simuliidae

Adult black fly

Hernández (2008)

Spreading board

Insect braced on mounting board

Drying  Room temperature  Drying box: 100 Watt lamp  Incubator: 40-50C

Preserving box  Creosote  P-dichlorobenzene

Wet preservation  Following fixation, the most commonly used preservative is ethyl alcohol in various concentrations.

 Aphids and scale insects be preserved in lactic-alcohol, a mixture of 2 parts 95% ethyl alcohol + 1 part 75% lactic acid.  80-95% ethyl alcohol for molecular studies  Isopropanol can be applied for preserving  Stored with a data label in a separate glass vial  Sealed vial to prevent evaporation Upton and Mantle (2010)

Preserving jar Storing small tubes containing specimens

 Microscope slide  Hoyer’s media, Euparol, Canada balsam

Larva

Larval and pupal exuviae

Pupa

Genitalia

Labelling  Pinning insects: white cards

 Insects preserved in fluid: white paper & pencil  Mounting slide

Mounting slide of mosquito larva THAILAND

Chumphon. 06.V.2018

Culex quinquefasciatus

Ban Kon Thae Coll: Name HBT: water jar Slide#F-8204

Det. Name

Pinning mosquito adult THAILAND: A2 BCLp12(1)-21 ♀ Lampang Province, Ko Kha District,

Ban Don Thum. Human-baited traps 23.XI.2013

Coll: Choochote et al.

Anopheles (Anopheles) saeungae

Det. Taai & Harbach 2014

Storage

Cabinet

Original photograph from Dr. Wichai Srisuka Entomology Section, Queen Sirikit Botanic Garden Chiang Mai, Thailand

Preferred methods of preserving insects and arachnids

Millar et al. (2000)

Additional Readings  Millar IM, Uys VM, Urban RP. Collecting and preserving insects and arachnids. A Manual for Entomology and Arachnology by SAFRINET, the Southern African (SADC) loop of bionetinternational.  Schauff ME. Collecting and preserving insects and mites: techniques and tools. Systematic entomology laboratory, USDA National Museum of Natural History, NHB-168, Washington, DC 20560.  Upton MS, Mantle BL. Methods for collecting, preserving and studying insects and other terrestrial arthropods. The Australian Entomological Society Miscellaneous Publication No. 3. 2010. pp. 81.