DIAGNOSTIC AND SAMPLE PROCESSING OF PLANT PATHOGEN, PEST,

PLANT SPECIMENS TRAINING WORKSHOP

INSECT PESTS:
Lepidoptera, Coleoptera, Hemiptera, Diptera and others

Hari Sutrisno & Darmawan

Research Center for Biology

The Indonesian Institute of Sciences

2013

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 Handling specimens of insect pests

(Lepidoptera, Coleoptera, Hemiptera and Diptera)

by

Hari Sutrisno

Zoology Division, Research Center for Biology – LIPI, Bogor, Indonesia

Jl. Raya Bogor Km 46, Cibinong, 16911. Email: [email protected]

1. Lepidoptera: moths and butterflies

Lepidos= scale, pteron= wing

One of the major orders in size, > 160 000 species (70 families)

Adults:

- small to large,

- hypognathous,

- nearly all with long coiled proboscis,

- antennae multisegmented and often comb-like (pectinate), clubbed in

butterflies

- wing with double layer of scales and large scales including the discall cell

Immature stages (larvae, caterpillars):

- with sclerotized mandibulate head

- labial spinnerets producing silk

- jointed legs on thorax, and some abdominal prolegs

Butterflies:

- day-flying,

- hold their wings together vertically above the body,

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 - antennae knobbed or clubbed,

Moths:

- active at night or dusk,

- hold their wings flat or wrapped around the body,

- antennae pectinate,

Their habitat:

- feed utilize nutritious liquids such as nectar, honeydew and other

seepages from live and decaying plants, and few species pierce fruit

- many species supplement their diet by feeding on nitrogenous animal waste.

- most larvae feed exposed on higher plants and form the major insect phytophagus,

- few primitive species feed on non-angiosperm plants, some on fungi;

- several are predators and others are scavengers, notably amongst Tineidae (wool moths)

2. Coleoptera: beetles

- about 350 000 described species in the world, more species than vascular plants or

fungi and 90 time as many as Mammals

- diverse in structure and size;

- the largest: Cerambycidae Titanus giganteus from south America and Xixuthrus heros

from Fiji, attain a length of 200 mm.

Adults:

- small to large, often sturdy and compact

- heavily sclerotized or armoured, mandibulate,

- fore wing modified as rigid elytra covering folded hind wings at rest

- legs variously modified, often with claws and adhesive structures

Immature stages:

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 - terrestrial or aquatic with sclerotized head capsule, opposable mandible

- usually 5 segmented thoracic legs, without abdominal legs or labial silk gland

Their habitats:

- vegetative surface (Forest canopy): Scarabidae: Melolonthinae

- flowers and Male Cones- Pollen-feeding

- cones, Seeds and Seed pods: Curculionidae, Anthribidae

- bark and Wood surfaces

- subcortical Habitats- Under bark of Living and dead trees and logs

- living and dead wood (wood borers): Lucanidae, Passalidae

- herbaceous plant tissue, stem borers, gall makers and leaf miners

- palm fronds

- leaf litter and ground debris

- animal nest

- caves

- grasses and gransslands

- soil

- dung

- fungal fruiting bodies

- marine and freshwater littoral zones

- standing freshwater littoral zones

- standing freshwater (lentic habitats)

- moving freshwater (lotic and hydropetric habitats)

3. Hemiptera:

Suborder: Homoptera (Sternorrhyncha, Auchenorrhyncha), Heteroptera

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(Greek: hemi = half; pteron = wing)

Common names: aphids, scale insects, white flies, leafhoppers, cicadas and bugs.

Distribution: Cosmopolitan

Hot to recognize (description):

The Hemiptera are the dominant group of exopterygota insects. They range in

length from less than 1 to 110 mm, and comprise insects with a great range of different

structural and behavioral features and occupying a wide variety of environment.

The most characteristic features is the structure of the mouthparts, which are

highly efficient for extracting the liquid contents of plants or animal prey. Bugs possess

piercing-sucking mouthparts in a simple tube (rostrum) formed by modification of the

insect’s lower tip (labium). Inside the tube, modified mandibles from canals which

allow an upward flow of liquid food and a downward flow of saliva. The modified

mandibles are called sylets. Often the rostrum is held under the head but is brought

forward during feeding. Compound eyes are usually present and well developed; simple

eyes (ocelli) may be present. Antennae may be short with only a few segments, or well

developed and more less filiform. Most species possess two pairs of wings, but some

have only one pair and a few species have none. The order name describes a character

of the many bugs in which the front pair of wings is modified so that the basal half of

each wing is hardened (sclerotic) to form a protective cover (a hemelytra). As the wing

bugs are folded flat on the abdomen, this makes the insect appear as if half of each of

forewings is missing. In the remainder of the order, the wings are held over the

abdomen rather like two sides of a house roof (cicada, etc.).

Most Hemiptera are terrestrial and phytophagous. In suborder of Homoptera, the

family Cicadellidae is a very diverse of group of Auchenorrhyncha, and the largest in

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number, more than 15.000 species described. Most species are phloem feeders and

important as vector of virus diseases. At species level, examination of the male

genitalia is essential.

In suborder Heteroptera (true bugs), include groups which are carnivorous and

often produce a repellent odour-used for defense from a specialized gland. Many of

which are also aquatic, such as water boatman (family Corixidae).

Members: Bugs, water strides, water scorpion, water bugs, water boatman, cicadas,

leafhoppers, spittlebugs, aphid, psyllids and whiteflies.

Habitat, Plant, Food: Bugs feed on liquid obtained from plant or animals including

man. Any part of the plant may used as food: leaves, stems, fruits or roots. Blood is

consumed by some species, while others consume insect body fluid.

Importance:

The plant feeding bugs are considered serious pests in agriculture such as brown

plant hoppers Nilaparvata lugens in rice plant and the green leafhopper Nephotettix

virescens, besides direct feeding also act as vector of virus diseases. Scale insect

infestations can seriously damage citrus crops and ornamentals, and aphid attack can

harm various ornamentals such as Macrosiphum rosae in roses.

The characteristics of Aphids: soft-bodied insects up to 4 mm long; most have a pair of

siphunculi/cornicles towards the posterior end of the abdomen and long antennae.

They often form large colonies on leaves and stems. In the tropics, about 30 species

are pests. . Many species have been implicated as major vectors of plant virus

diseases, but damage by sap depletion, saliva toxicity and sooty mould growth can also

be serious.

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4. Diptera: Focused on Fruit Flies

(Greek, di=twice, pteron=wing)

Common Names: flies, crane flies, mosquitoes, midges, sandflies

Distribution: Cosmopolitan

Suborder: Nematocera and Brachycera

How to recognize (description):

The Diptera form one of the larger insect order, the world total of species,

described and undescribed, probably being at least 150 000. All winged species of

flies are distinguished by having only one pair of functional wings. The members of the

order have a pair of membranous forewings used for flight while the hind wings are

modified to form short, club-like structure called halteres. Halteres are used as

balancing organs during flight. Some dipteran species are wingless throughout their life

cycles and their position within the order must then be decided on using other aspects of

their structure and life cycle. Well developed compound eyes are present. The adult

body is often hairy and depending on the species, hairs maybe few or abundant, short or

long. Adult mouthparts are typically modified for sucking or sponging up fluid, but may

also be adapted for piercing. Dipteran mouthparts are complex structures composed of a

number of parts (e.g. labium, stylets, labellum, etc.).

All members have a metamorphic life cycle: egg-larva-pupa-adult. The larvae of

most flies are legless maggots. The free-swimmong mosquito larvae are called wrigglers

and these posses a well defined head and mandibles;

Members

Fruit flies, crane flies, blow flies, bee flies, dung flies, house flies, bush flies,

robber flies, march flies, vinegar flies, mosquitoes, midges, gnats.

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Habitat, plants, food

Flies and mosquitoes feed on wide variety of materials mostly involving fluids e.g.

blood, dung fluids, piant or animal body exudates. When solids are consumed the flies

often use saliva to partially digest or disoolve the solid before it is lapped up by the

proboscis (mouthparts)

Importance:

Flies and mosquitoes are one of the most successful insect orders with perhaps

250.000 species. They are immensely important as transmitters of disease and parasites.

Human diseases include: sleeping sickness (tse-tse fly); malaria, filarial, yellow fever,

dengue fever, Murray valley encephalitis (mosquitoes); dysentery, opthalmia, cholera,

and typhoid fever (house fly).Millions of dollars worth of damage to fruit crops

(together with equally large costs of sprays to protect the crops) are caused by fruit flies

of various kinds. Blow flies, screwworm flies and buffalo flies attack open wounds on

animals, burrow into the animal tissues or suck blood from the animal. This requires

expensive methods to combat their predations.

Collecting methods:

Hand collecting

Sweeping

Beating and Brushing

Light trap

Litter extraction: Barlese

Sifting litter

Pitfall Trap

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 FIT (Flight Intercept Trap)

Malaise Trap

Canopy fogging

Hand collecting

Hand collecting is still the best way to capture insects within their

microhabitats and often to obtain living adults, especially for beetles. Usually we use

forceps to collect the large one, but for collecting small and active specimens we use

aspirator. This method is not suitable for the adults of Lepidoptera and wasps.

Sweeping

Sweeping may be the most convenient way to collect day flying, crepuscular or

very small species and species not attracted to light (Butterflies). This method is also

designed to collect surface active insects from vegetation and also very efficient for

collecting in grassland and among herbaceous plants.

A heavy gauge insect net is literally swept through the vegetation and the

insects collect at the bottom along wit bits of vegetation. The collected debris may be

spread out on a tray or placed in a container and sorted latter in the laboratory and we

use an aspirator to collect small insects such as parasitoid wasps or small beetles.

For collecting butterflies, we use a light and smooth sweeping net with a long

stick to catch them at the canopy or when they are flying.

Light collecting

Light collecting is very efficient in collecting insect attracted to light.

Collecting at bright lights for Coleoptera is not as productive as it is for Lepidoptera,

but on warmer nights large numbers of beetle may be obtained in this way. Mercury

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vapour lamps or UV fluorescent tubes are the most attractive to insect. Adult of many

Carabidae and Staphylinidae, water beetle, such as Dytiscidae, Hydrophilidae and

Heteroceridae and various Cerambycidae and Curculionidae can be found at this light.

There are two methods; using a light sheet and using light trap. With a light

sheet a white sheet is suspended beside a light source and moths are selected as they

come to rest on the sheet. Only specimens required need be killed but the sheet must

be attended by the collector. Most often we use a 160 watt (mercury vapour plus

tungsten filament) blended lamp run off mains power or generator. We also use a 250

watt mercury vapour lamp with a ballast also run off mains power or generator. Care

must be taken with MV lamps as some can cause sunburn or eye damage. The higher

powered lamp tends to attract higher flying moths but may be too bright for forest floor

species which may settle a long way from the light. In the USA black light tubes run

from a car battery used.

Light traps are usually run unattended which means many units can be run

simultaneously. They kill may not target insects and the operator usually has more

exposure to dangerous chemicals. There are many designs for traps and they may be

designed to collect, for example, small moths or to exclude beetles. We use a large 80

watt mercury vapour light trap run from mains power or a generator and small 8 watt

black light tube traps run from a 12 volt motorcycle battery. Material in reasonable

condition for identification can usually be obtained with the use of tetrachloroethane as

a killing agent. Material from traps without a killing agent is usually unidentifiable.

Self-sorting light trap also can be used to collect insects. The trap design is

based on the fact that heavier insect tend to fall a crawl around when the strike the vanes

of the trap, while the smaller more fragile insects to fall more gently and fly again

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 immediately on landing.

The self-sorting container collecting container consisting of a deep stainless

steel tray inside of which fits two removable trays each divided into sections, and top of

which fits stainless steel lid with a central square hole and corner lugs into which the

legs of the funnel assembly fit. There is steel plug that seals the square hole.

The lamp and funnel assembly consisting of a 125 watt mercury vapour lamp

surrounded by four stainless steel vanes and mounted centrally over a stainless steel

funnel. This unit, which is supported on four legs, is protected from the weather by

transparent polycarbonate roof fastened to the top of the vanes.

Light collecting is affected by the following factors:

1. Phase of the moon. Moths are less active in bright moonlight. The effective

radius of a lamp is reduced by “competition” from moonlight. For the best results

trapping is best carried out from the fourth day after full moon until about one week

before next full moon. The trap should be placed in a sheltered position, and tracks, or

similarly small, cleared area, within forests should be chosen.

2. Temperature and humidity. Moths are very sensitive to temperature and are much

less active in seasonally cold conditions. Moths tend to be more active with high

humidity.

3. Wind. Moths fly less in windy conditions and those attracted may not land at the

light. Moths settle out of the wind and fly upwind when possible so the light must be

on the sheltered side of the sheet and the sheet or trap must be in as sheltered a place as

possible.

4. Light characteristics. Moths are, in general, most responsive to light in the near

ultraviolet and blue part of the spectrum. Light with high output of wavelengths at

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 about 380 and 460 nm are effective.

5. Lamp power. Higher powered lamps will draw moths from a longer distance or

from higher but many moths will settle before coming to the sheet. Higher power

lamps are probably best for large high flying species but lower powered lamps are

probably better for smaller species.

Moths collected at the sheet can be transferred to a killing bottle in glass tube

or frozen immediately in the tube. Moths killed in a trap should be sorted while fresh

and without drying out and those required pinned. Those discarded should be buried

as birds may be poisoned by moths containing tetrachloroethane.

Beating

This method involves the beating of branches and shrubs with a stick in order

to dislodge surface active insects. Specimens will fall on the flat surface of a beating

tray which consists of a piece of heavy cloths spread out on a wooden or metal frame.

They specimens may be hand collected with forceps or pick up with an aspirator.

Litter extraction: Barlese (Tullgren) funnel.

In this method, the litter is placed on a fine grid and either subjected to slow air

drying or heated more rapidly by means of a low wattage light bulb placed directly over

it. Arthropods and other organisms move away from the heat sources, dropping trough

the grid and into the collecting jar (usually containing ethanol).

Sifting litter

This method is suitable for collecting small beetles that live in the ground.

This sifter consists of a heavy cloth cone about 120 cm in length, 30 cm in diameter at

one end and 10 cm at the other. An open metal frame with a handle attached is sewn

into the large end and another similar frame, to which a metal 1 cm grid is soldered,

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attached about 25 cm below the first one. The narrow end of the cone is tied shut with

a rope, so that a bag is formed. Litter or debris placed in the top of the bag rest on the

grid, and we shake the sifter, fine debris, including small arthropods falls through the

grid and accumulate at the bottom. Then, using a tray for spread out the debris and

using aspirator to catch the small beetles.

Pitfall Trap

This trap consists of plastic glass or metal container placed into the ground so

that their open ends are flush with or slightly below ground level. This glass usually is

filled with a variety of collecting fluids, such as ethanol, detergent and water or

propylene glycol. In order to keep rain water from filling the pits, they are usually

covered with some kind of roof. Small organisms moving on the ground will fall into

the pit.

FIT (Flight Intercept Traps)

Flight intercept is used to catch flying insect that have tendency to drop when

intercepted flight. These are collected in a trough set into the ground below the

intercept pane. The trough, if set with its top level with the ground surface, also acts

as pitfall trap for ground dwelling arthropods.

They consist basically of an intercept pane of fine black material strung

between two poles and suspended above an open trough set in the ground with its top at

ground level. A black plastic roof is provided above the intercept pane as a rain

excluder. The accompanying rough diagram will serve to show a trap set up for

operation.

This type of trap can be left in place for many months and they have proved to

be very productive over periods of up to several years. Organisms in the trough may

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 be regularly collected and the level of liquid in trough maintained.

Materials:

1X plastic gutter trough with ends (1.5 m) (made from 150 mm stormwater pipe

capped at both ends and cut in half longitudinally.

1X Fine mesh intercept pane with slots sewn at either end to take poles (= 1.00 m

high x= 1.7 m long).

2X Poles (= 2 m)

1X A piece of strong black plastic double to a size of = 2.5m X 1.5m

Ropes

Collecting/ Preserving liquid (for trough of 10 L capacity):

6 liter of water

4 liter of propylene glycol (40%)

100 ml propylene phenoxytol (1%)

100 ml glacial ecetic acid (1%)

Sampling Equipment:

1X fine mesh aquarium fish net (12 cm or smaller)

several wide mouthed jar with lids, filled with 80% ethanol

1X squirt bottle filled with 80% ethanol

80% ethanol

Labels

Set up Procedure:

1. Select a suitable site. This might be rainforest, open forest or more open country

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 but remember that the traps may be in place for a year or more. Small trees to attach

the ropes to would be an advantage.

2. Dig a trench for the trough. Make sure that it allows the trough to be as near to

horizontal as possible, otherwise the liquid in the trough will be of unequal depth

throughout. Place the trough in the trench so that its top is level with the ground

surface. Backfill enough earth to stabilize the trough and provide a natural lead up to

the trough for ground dwelling arthropods.

3. Take the intercept pane and place the poles in the slots at either and. Now locate

the poles at either end of the trough so that the intercept pane will be centered along the

trough. Hammer poles into ground, making sure that the pane remains taut, especially

the lower end above the trough. Secure the poles by tying with ropes to any suitable

object.

4. Place the double thickness plastic roof over the top of the poles which have

previously been padded to prevent ripping the plastic. Secure ropes to each corner,

using small stones as anchor points in the plastic. Secure roof to top of each pole by

pushing down and tying. Secure each corner rope to any suitable object so that the

roof is reasonable taut.

5. Remove any loose dirt, sand or debris from the trough and fill with the various

components of the preserving liquid to just below the top, being sure to mix it

thoroughly with a stick or other suitable object.

6. Collect any arthropods at approximately 3-4 weeks intervals and check on the level

liquid in the trough.

Sampling Procedure:

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1. Remove any large pieces of debris as leaves and twigs, making sure to wash them in

the trough or liquid to remove any adhering arthropods.

2. Take the fish net and collect all the arthropods in it using a backward and forwards

motion along the trough. Empty into the wide necked jars of 80% ethanol, using the

squirt bottle of 80% ethanol to help if necessary. Repeat procedure until all arthropods

are removed.

3. Make label (s) and place in jar(s). Strong paper with information in lead pencil is

suitable.

4. Check the level of liquid in the trough and top up with water if necessary together

with extra propylene glycol, and glacial acetic acid in appropriate quantities. Add

extra propylene phenoxytol and squirt of detergent every other month. The propylene

phenoxytol can be omitted if in short supply or if too expensive.

Canopy fogging

Fogging with insecticides is the most recent and the most rewarding of the

mass collecting techniques. Usually pyrethrin-based insecticide is used for spraying

the canopy foliage. The insect will drop into spreading sheets on the group. This

method is often to be used for counting the diversity of a certain area.

How to collect fruit fly

Fruit fly B. dorsalis spp. are very serious pest of a wide variety of fruits and

vegetables throughout its range and damage levels can be anything up to 100% of

unprotected fruit. Eggs of B. dorsalis are laid below the skin of the host fruit. The

eggs hatch within a day (although delayed up to 20 days in cool conditions) and the

larvae feed for another 6-35 days, depending on season. On fruit attacked by fruit fly,

there may be some necrosis around the puncture mark ('sting'), and then is followed by

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decomposition of the fruit. Detection can be done by split the fruit to collect the larvae.

Identification based on larvae rather difficult to be done. That why, the larvae should

be reared in saw dust in cages to be pupated and emerged as adult.

In the field, pupation is in the soil under the host plant for 10-12 days but may be

delayed for up to 90 days under cool conditions. Adults occur throughout the year and

begin mating after about 8-12 days, and may live 1-3 months depending on temperature

(up to 12 months in cool conditions) (Christenson and Foote, 1960). Adult can be

collected using methyl eugenol or cue lure trap for attracting males of Bactrocera spp.

Plant parts liable to carry the fruit fly in trade/transport:

Adult flight and the transport of infected fruit are the major means of movement and

dispersal to previously uninfested areas. The major risk is from the import of fruit

containing larvae, either as part of cargo, or through the smuggling of fruit in airline

passenger baggage or mail. Individuals who successfully smuggle fruit are likely to

discard it when they discover that it is rotten.

Preservation

Killing

For moths, there are 3 main methods. Freezing, use of killing bottles (KCN or

Ethyl acetate) and tetrachloroethane. For butterflies, we can press the thorax by using

our fingers gently for few seconds. For beetles and Wasps, we can use killing bottle

(ethyl acetate).

Freezing is the best to kill moths where a freezer is available. Specimens can

also be stored in an airtight container as long as they do not rattle around. They can be

pinned and set as soon as they thaw.

Killing bottles are convenient for individually collected specimens where a

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freezer is not available. They can be made from KCN in which in case the moisture

level is critical, moths must not get wet and must not dry out. Ethyl acetate is also

effective and is more readily available.

Tetrachloroethane is the standard killing agent in light traps. It knocks down

quickly and the vapour is much heavier than air but it is slow to kill. It is highly toxic

to humans and is absorbed through the skin. It has a distinctive smell and inhalation

can be largely avoided. It must be allowed to disperse before the catch is stored.

Pinning

All killed specimens should be pinned. Never place moths in envelops.

Never allow moths to rattle around in a tube. Moths must be fully relaxed when

pinned and should be pinned as soon after death as possible.

Macros are pinned vertically through centre of the thorax with a 38 mm

stainless steel pin and the moth should rest 2/3 of the way up the pin from the tip. We

usually use no 3 size but a no 1 size can be used for small bodied moths but in stiff cork

the pin is more easily bent.

Micros should be pinned vertically through the thorax with a 15 mm 0.01 diam

micropin, again the moths should be about 2/3 of the way up the pin. The moth should

be well relaxed. Very small moths require even smaller and finer pins. Great care to

place the pin vertically is repaid by subsequent ease of handling.

Temporary storage

Macro moths are best stored pinned in a storebox. Moth should never be

stored in paper envelopes as appendages break off, they are difficult to examine and

difficult to relax and set later.

Micro should be pinned into a shallow tray lined with plastic foam and the wings lifted

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away from the body into a semi set position. This facilitates later examination and

setting.

It is usual to bulk label specimens in temporary storage. Unless frozen or set fresh

moths should be dried rapidly in storage.

Relaxing

Moths too dry to set but which need setting must be relaxed. A relaxing jar

contains a layer of boiled and cooled water or damp sand and sometimes some

antifungal agent, thymol or chlorocresol. Moths are pinned into foam in the saturated

atmosphere above. Absorbent material draped around can help increase the surface

area of the water. Time taken to relax depends on the size of the specimen, a little on

its age and a lot on the temperature. At a constant 21 C we leave large moths relaxing

for 18-24 hours. They are not then properly relaxed but the appendages are. Large

moths are then injected in the thorax, using a hypodermic syringe, with a small amount

of hot water. This requires practice as the moths must not become wet. After 10-15

minutes the can be set. Smaller macro moths can be set after 18-24 hours without

injection. Micro moths relax in 4-6 hours and can than be set. Very small ones are

even quicker. Relaxing jars are very sensitive to temperature and the degree of

airtightness. Practice with your own set up is necessary.

There is special technique to relax the betel using beetle relaxing fluid which is

developed at the United National Museum by H. S. Barber and others. One formula

which has been used with considerable success is given as follows:

Alcohol 95% ………………………………..265 part

Water (distilled) ……………………………245 part

Ethyl acetate ……………………………… 95 part

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 Benzene ……………………………………. 35 part

If, when making up, the mix should separate into two layers or the mixture

remains cloudy, absolute alcohol may be added, a few drops at a time, with gentle

shaking until the fluid is clear.

Beetles killed in this fluid are relaxed completely and the genitalia extruded

after soaking for about twelve hours. Specimens may be pinned immediately on

removal since evaporation is rapid and setae, pubescence, antennae and other small

appendages spring readily back into position. Those requiring to be mounted with

adhesive are best left until dry. Colours are not affected.

It has also been found that the fluid is invaluable for rejuvenating old

specimens. Soaking for half an hour relaxes dry specimens sufficiently for handling,

resetting or repining. Light mould patches and grease are removed and most mounting

adhesives in common use are softened or dissolved. Individual limbs or joints can be

loosened to allow movement without injury by the local application of a single drop of

fluid. This is sometimes necessary when pinned specimens are being examined.

The most usual application is to coleoptera, but orthoptera, hemiptera, diptera

and hymenoptera have also been relaxed by the fluid, although care had to be taken that

the edges of wings did not fold over. It has been used successfully on phorids and

drosophilids but flies smaller than these and tiny hymenopterans fold up while being

removed from the liquid and are difficult to straighten out when dry.

Setting

Numerous account of the process are available from the references. There are

some critical points.

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Moths, even fresh ones, must be thoroughly relaxed first.

The distance from the tip of the pin to the top of the board, (the height of the specimens)

should be standard. We use about 24 mm for macros. This should leave space for the

use of forceps to handle the specimens above and space below for legs and labels.

The moth when placed in the groove must be correctly oriented and the groove

the correct width. The body should be clear of the sides but only by 1 or 2 mm. The

body must not slope up or down or be higher on one side. If not correctly placed great

difficult will be experienced in adjusting the wings.

Moths must dry thoroughly on the boards. We leave them 4 weeks which is

probably unnecessarily long. If you are without humidity control do not remove from

the boards in wet weather. If possible specimens should be individually labeled when

set. If permanent labels are not yet available then a foolproof system of temporary

labels is needed. Never use numbers or roman numerals for months on labels. Spell

the month out or use the first three letters.

Staging

Micro moths, on 15 mm pins, when removed from the setting boards must be

staged. This is necessary because the fine pin needed for small moths would bend too

easily if it were long enough to take the labels often needed. A block of polyporus pith

of fine white plastic foam 12 mm long (sometimes 14 mm for larger moths) is pinned

2/3 of the way up a 38 mm No. 3 pin. The micropin is inserted in this so that

specimens can be handled by the large pin and labels placed on the large pin. The tip

of the pith can provide some protection for the head of the moth.

Labeling

The value of any specimens is in direct proportion to the completeness of the

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data on the label attached to it. Label should be entirely adequate but concise; they

should be accurate, unambiguous, legible and permanent. It should be remembered

when accessing specimens that they may be examined later by research workers from

throughout the world whose knowledge of the language used may be poor and the

local geography nil. Furthermore, the specimens will be available for many

generations to come.

The minimum label data required with specimens are LOCALITY, DATE and

COLLECTOR’S NAME.

Storage

Cabinet drawers offer better storage than storeboxes as moths can be examined

without opening. Properly designed cabinets can be pest proof and contain a pest

repellent. It is very useful if the collection area is air conditioned and temperature and

humidity controlled. Cooler temperatures provide better very long term storage but

comfortable working conditions are necessary. Humidity control helps control pests,

particularly mould, and also helps to prevent set specimens from “springing”. In our

museum temperature is controlled at about 21 C and the relative humidity at around

50%. This eliminates problems from mould and psocids and naphthalene in sealed

cabinets largely prevents Anthrenus damage.

The use of unit trays is universal in the Lepidoptera unit except in those species

which are so large that only 5 rows can fit in a drawer such as Saturniidae, large

Hepialidae, and large Cossidae. The advantage of being able to rapidly recognize a

drawer shuffling unit tray is great even though fewer specimens can be put in a drawer

When using unit tray each drawer must be filled with trays. Loose trays with

specimen will slide about when drawers are opened and closed, badly damaging the

22
specimens.

Lepidoptera fade rapidly (particularly moths) in strong white light. Specimen

for long term preservation should not be displayed more than necessary.

Degreasing.

Some moths, Cossidae, some Hepialidae and Lasiocampidae become greasy as

body fast break down. Usually this is most serious in male. In small specimens a

piece of curled blotting paper may be pinned beneath the moth in contact with the

abdomen and this soaks up much of the grease. With large Cossidae and Hepialidae

the moth may be bathed in a bath of ethyl acetate for a week and pinned to dry on a

wide setting board. When dry the scales can be fluffed up with a brush so that the

moth does not look as if it has been wet. Greasiness can be prevented by degutting the

moth when fresh but this destroys the internal and often the external genitalia which are

taxonomically important. For this reason we never degut moths.

How to preserve the Diptera

Fix larvae in Carnoys or 990 C water, preserve in 80% ethanol. Do not set wings of

species smaller than blowfly size but arrange wings up and away from body and extend

legs downward. Pin adults through mesothorax slightly to right of midline. Set wings of

large species so that wings are anterior to the right angle position. Stage smaller species

on pith. Very small species should be glued to apex of card triangle.

Adult craneflies (Tipulidae) may be preserved in 80% ethanol or glued to a

rectangular card by side of thorax with legs secured to card by a spot of glue near apex

of each tibia.

Biting midges (Ceratopogonidae) should be killed and preserved in 80% ethanol

Preservation for Hemiptera:

23
 Kill and preserve nymphs in 80% ethanol. Most Hemiptera adults are preserved

dry on pins or points. Pin large Heteroptera through the right side of scutellum

(hemelytron); care must be taken in pinning not to destroy structures on the ventral side

of the thorax that will be used in identification. Pin medium sized species through base

of right wing. Very small Heteroptera may be glued to apex of card triangle with its tip

bent down.

Set left pair of wings of Auchenorrhyncha with anterior margin of hindwing at

right angles to body and forewing immediately in front. Most Hemiptera, less than 10

mm in length should be mounted on points, or should be stages on micropin and pith or

glued to apex of card triangle with specimen lying on its left side. Specimens mounted

on points should be mounted so that the beak, legs, and ventral side of the body are not

embedded in glue. If specimen is mounted dorsal side up on the tip of a point, the point

should not extend beyond the middle of the ventral side of the insect.

Kill and preserve Stenorrhyncha in 80% ethanol. Scale insect may be preserved

dry on host plant and placed in vial.

Killing bottle made for most Hemiptera is a small bottle or larger bottle depend

on the size of insect to be killed, at the based poured with sodium or potassium cyanide,

finely granular or powdered form with wet plaster. After the wet plaster has been poured

in, the bottle should be left uncorked a day or two, until the plaster has thoroughly set

and dried, then it is corked. A poison label is put on. Another material that can be used

as killing agents in insect bottles is ethyl acetate, carbon tetrachloride and chloroform.

Ethyl acetate is the least dangerous of the three to use.

Mounting on microscope slides

Many small arthropods (thrips, lice, fleas, mites, aphid) or isolated body parts as

24
genitalia are best studied when mounted on microscope slides. Material so mounted is

generally transferred to a slide from preserving fluid, and the mount may be temporary

or permanent. The media for temporary slides mounts are glycerine, and for permanent

slides mount the most commonly used resin is balsam. Specimens mounted in a resin

must first be dehydrated (by running through successively increasing concentration of

alcohol: 70, 95, 100 %)., and then through xylol and into resin. For dark colored or thick

bodied specimens or such structures as genitalia, must be cleared before mounting.

Several substances used as clearing agents is potassium hydroxide (KOH), that

can be used for almost any arthropod or arthropod structure. After clearing in KOH, the

specimens should be washed in water (preferably with a little acetic acid added) to

remove any excess of the KOH. KOH can be used cold that the clearing may require

from a few hours or warm, the specimen may be boiled in few minutes. It is sometimes

desirable to stain an insect before it is mounted, very commonly used is acid fuchsin.

Material for identifications

This should be pinned as outlined above. It must be pinned but it does not

need to be set. Set material is easier to identify but unless it is set expertly it is better

left unset. It must be fully labeled with as much information as possible. If possible

all available material should be included, often one sex is easier to identify than the

other and sometimes a second, unexpected, species is present.

It is very hard to convince a taxonomist that an identification is really needed if

little trouble is taken to prepare the material or if little material is available of a claimed

pest. Well prepared material can usually be identified, as far, as possible, quickly and

accurately.

Packing

25
 Moths should be pinned and crosspinned in a strong cardboard box packed around with

woodwool, foam beads or crumpled newspaper and placed within a larger, strong

cardboard box. The packing should be firm, not loose and not tight. Heavy moths

must be extensively crosspined to prevent any movement. Never place microscope

slides or naphthalene in the same box with insects as they frequently come loose and

cause great damage.

Mailing specimen

The following point should be observed when sending specimens through the mail.

Avoiding damage:

Many specimens reach their destination in broken or irretrievably damaged

condition. Nearly always this is because of poor packing of specimen before dispatch.

Damage can be avoided by taking the following simple precaution:

1. Specimen in fluid.

These should be mailed in screw-cap vials, which are usually leak-proof even if not

taped. Vials with press-in or press-over caps are not satisfactory and corks, if used,

should be waxed for leak-proofing. Glass vials are safe as plastic vials when properly

packed. Each vial should be individually wrapped in tissue or cotton-wool packing.

After individual wrapping vials need to be placed in a strong container such as a

wooden box or tin, and this in turn packed in a large carton containing shock-absorbent

material such as cotton-wool, polystyrene chips or wood-wool; for individual vials

specially hollowed wooden containers are available and are ideal. Each vial should be

lightly plugged internally with tissue or similar material to prevent specimens from

slopping back and forth in transit, and air-bubbles should be rigorously excluded (many

fluid-preserved specimens reach their destination in a damaged or part dried condition

26
 because steps have not been taken to prevent swilling and eliminate air).

2. Pinned adult

They should be securely pinned into a rigid box of wood or stout cardboard or plastic.

If the specimens are staged the mounts should be cross-pinned to prevent them swinging,

or if not cross-pinned then the specimens positioned in the box, so that they could not

touch one another if the mount rotated. The specimen-box should contain a corner

piece of pinned cotton-wool to catch any specimens or structure that breaks loose.

3. Slide mounts:

These are the best sent in a purpose-built slide box with spacers to hold the individual

slides in place. A thin layer of soft packing should be placed underneath and over the

top of the slides. Plus all empty space in the box should be filled with soft packing.

Finally the lid should be taped firmly in place. You can check the packing by listening

to see if the slides move around inside when the box is shaken.

4. Packaging the specimen container:

This container should then be packed into a much larger carton so that there is a

minimum of three-inch thickness of soft packing material (e.g polystyrene beans or

wood-wool) around each surface of even a small specimen-box – and more than this for

large store boxes. Mail office requirements vary, but packages should carry “fragile”

stickers and labels indicating the contents as “specimens for scientific study: no

commercial value”.

5. Postage

If possible specimens should always be sent by airmail. Also the package should be

registered.

27
Techniques for collecting, rearing, preserving immature insects (Lepidotera,

Coleoptera, Hemiptera and Diptera):

Collecting

Immature insects can be found everywhere except in the air and in

salt water. Many caterpillars, and some betel larvae feed externally on plants and are

easily collected by: Sweeping, beating, or searching for

damaged or webbed parts of plants.

Many other immatures are borers in all parts of living, dying, dead, decaying

and decayed plant parts. Borers are commonly indicated by dead twigs, buds, and

cones, damaged fruit, exuding sap, and accumulation of frass. They can be collected

by peeling, prying, splitting and and chopping the wood in apart.

A great variety of immatures inhabiting ground litter and soil can be collected

with anything from a hand trowel to a plow, but other more specialized and labor-saving

methods such as Barlese or Tullgren funnels and pitfall traps are very effective for many

groups.

Parasitoid larvae (wasps) are relatively easy to collect by dissecting or holding

the host, but because identification even to family may be difficult, rearing to adults is

the best way.

Rearing

Adult is very important in classification since some immature stages of insects

are unknown. Reared material is essential for the advancement of our knowledge of

immature insects. Natural enemies may also be reared; they should always be saved

and properly labeled to associate them with the immatures they emerged from.

Although rearing may seem simple, quite often it is not, because the proper

28
food, temperature, humidity, and pupation site must be provided in a relatively confined

condition in the lab or in the field. So, the more mature the larvae are the more

successfully they are reared, but the early instars are obviously missed if only the

mature larvae are reared.

Ideally some specimens of all stages are preserved and some are reared to

adults, but problems arise when only one specimen is collected. Obviously it can not

be preserved but should be photographed if possible. In addition the cast skin should

be preserved (in 70% ETOH) for later examination since all external features will be

present and the skin can be softened for study or slide mounting.

Polyethylene bags can be used for most groups in many different ways.

Polyethylene is ideal in many respects because it is light, compact, cheap, waterproof

and disposable, but gases such as O2 readily pass through it. Leaf-feeding insects can

be maintained on cut foliage for several days in polyethylene bags, and rotten wood,

fungi can be kept moist.

It is essential to maintain proper temperature, moisture and humidity. Too

much water is as harmful as too little. Many larvae that live in soil, litter, logs, fungi,

stems, under rocks and in similar places are living under saturated or nearly saturated

atmospheric conditions. In the field, if conditions become unfavorable, they will move

toward more optimum condition. But at the laboratory it will not happen.

Proper food is essential; the obvious choice is the same food larvae were

feeding on when collected.

Preservation

The proper killing and preservation of larvae are very important. Most

Coleoptera, Lepidoptera and Hymenoptera larvae are not satisfactory preserved if

29
pinned and carded. Lepidoptera pupae can be pinned after drying.

In general, we used hot water or chemical agents to kill and preserve insect

larvae. Hot water is one of the best, all-purpose, killing agents because it stops

enzyme activity, thus killing the larva, and also allows it to be cleared of debris. We

can drop the specimens into boiling water and left to cool. Specimens are not usually

damaged by gently boiling, but some may burst, especially the larger, soft-bodied ones.

Some larvae of Scarabaeidae may darken after they are placed in alcohol unless they are

boiled for a minute or so.

We can also drop the specimens into cold water and heated to boiling. Small

amounts of ethyl alcohol can be added to enhance relaxation and straightening out.

The larvae may then be transferred to warm Kahle’s or Pampel’s solution which

promotes better fixation of the internal tissue. Large larvae should be pin punctured at

one or more place for rapid penetration of fixing agents. Most larvae killed in hot

water are preserved best in 75-80% ethyl alcohol, and should not be placed in 95% ethyl

alcohol which causes their collapse.

Beside of using hot water, we can also use chemical killing agents (Cold killing

solution) to kill larvae. KAAD is one of the most popular used. KAAD solution was

developed by Peterson in the 1940’s for field use. It was originally composed of 1 part

kerosene (K), 7-10 parts ethyl alcohol (A), 2 parts glacial acetic acid (A), and 1 part

1,4-dioxan (D). Various modification have been suggested by other workers, primarily

concerning the replacement of dioxin with ionic detergent (D) or other alcohols, since

dioxan function was to make the kerosene miscible with the alcohol. Dioxan presents

some risk of explosion if stored in a pure form for longer than 12 months. Ethyl

alcohol (95%) is used in KAAD, but other alcohols such as isopropyl have been used.

30
KAAD can not be made with 70% ethyl alcohol because of the miscibility problem with

the kerosene. Isopropyl alcohol is more miscible with kerosene than ethyl alcohol, and

can be substituted if emulsifier is not available. However, it does not penetrate as well,

or as fast, so specimens may have to be left in longer, but it does not burst larvae are

readily as ethyl alcohol.

Specimen should be put in KAAD while alive, since dead specimens usually do

not distend satisfactorily. They should be left in KAAD until fully distended; this may

vary from a few minutes to half an hour or longer, depending on their size and condition.

Specimens should be removed within 24 hours, or sooner if clearing or separation of the

cuticle occurs.

For permanent storage, the specimen should be transferred into 70-80% ethyl

alcohol. Due to the body fluids dilute in the alcohol, the alcohol should be changed at

least once before permanent storage, especially for large specimens.

Another chemical agent which is suitable for immature stages of wasps and

bees, some Trichoptera and Diptera is Kahle’s (also called Dietrich’s). This solution is

good fixative for internal tissues of both immatures and adults.

Formula for KAAD (Shake these until the solution is uniform and clearly).

Components Parts

Kerosene (coal oil) 1

95% ethyl alcohol 10

Glacial acetic acid

And either 2

Ionic detergent emulsifier 0.2 or more

(“Triton X-100”, “Tween”, or “Lubrol (x, w)”

31
 or

Isobutyl alcohol or

Secbutyl alcohol 0.5

Formula of Kahle’s solution:

Ethyl alcohol 95% 15

Distilled water 30

Formalin(40%) 6

Acetic acid, glacial 1

Slide preparation

It is necessary sometimes to examine detailed chaetotaxy or other minute

structures which are not visible on the whole larvae. In such instances, it is necessary

to clear the larva and make a microscope slide mount for detailed examination using

transmitted light.

The preserved larva is held with fine forceps and the head carefully removed

using a small, scalped blade. The larva is held on its left side and a slide made on the

right side above the thoracic legs, extending laterally to the apical abdomen segment.

The tip of the scalped blade is inserted into the abdomen and more cuts made in a

similar position until eventually the tenth abdominal segment is reached.

The body and severed head are then cleared by placing them in a 10% KOH

solution overnight or heated at 60-80 C on a hot plate for 20-60 minute until the muscle

etc are removed. Then placed them in distilled water on a microscope slide and

carefully remove muscle, fat etc.; for the entire body, the inside is best flushed out.

Then transfer to 70% ethyl alcohol for clearing.

Staining shows up structures such as weakly sclerotized tubercles, spiracles,

32
and setae. Stains such as 1% mercurochrome (in water) or carbol fuchsin (buy

prepared, or dissolve 4 gm fuchsin and 8 ml phenol in 20 ml 95% ethyl alcohol, and add

100 ml water) have worked well. Transfer the skin to the appropriate solvent for the

stain to be used, for example add enough Chlorozol black crystals to 70% ethyl alcohol

(not 95% with cause precipitation) to form a deep blue solution. Stain the skin for

5-30 minutes, or longer, until it is evenly stained. Do not overstain, since the skin will

not destain in alcohol. Transfer to 70% ethyl alcohol for rinsing. Skin can be stored

in microvials with other larvae, since the do not destain in 70% ethyl alcohol.

References

Common, I. F. B. 1990. Moths of Australia. Melbourne University Press.

Common, I. F. B. and Waterhouse, D. F. 1981. Butterflies of Australia. Angus and

Robertson.

IBOY-DIWPA. 2005. International field biology course in Indonesia: Coleoptera.

Cibinong, January 24th – February 2nd, 2005. Cooperation between Research

Center for Biology and The center of excellent program (Kyoto University,

Hokkaido University, Kanazawa University).

LaSalle, J and Gould, I. D. 1993. Hymenoptera and biodiversity. CAB International.

Lawrence, J. F and Britton, E. B. 1994. Australian Beetles. Melbourne University

Press.

May, B. M. 1938. Beetle relaxing fluid. The New Zealand Entomologist. Vol. 2:

2.

Stehr, F. W. 1987. Techniques for collecting, rearing, preserving, and studying

immature insects. In. Stehr, F. w (ed). Immature insects. Kendall/Hunt.

Publishing Company, Iowa.

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Upton, M. S. 1991. Methods for collecting, preserving, and studying insects and

related forms. Australian Entomological Society.

Weir, T. A. 1995. Set up sampling procedure for flight intercept traps. ANIC

Technical Training Course. Unpublished.

34